Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 208
American Journal of Biomedical Sciences
ISSN 1937-9080
nwpiicomajbms
Nox2ds-Tat A Peptide Inhibitor of NADPH Oxidase Exerts Cardioprotective
Effects by Attenuating Reactive Oxygen Species During IschemiaReperfusion
Injury
Qian Chen Issachar Devine Sydney Walker Hung Pham Regina Ondrasik Harsh Patel
William Chau C Woodworth Parker Kyle D Bartol Shayan Riahi Ashita Mittal Robert Barsotti
Lindon Young
Department of Bio-Medical Sciences Philadelphia College of Osteopathic Medicine 4170 City Avenue Philadelphia
USA Corresponding Author
Lindon Young PhD
Department of Bio-Medical Sciences
Philadelphia College of Osteopathic Medicine (PCOM)
4170 City Avenue
Philadelphia PA 19131
USA
Tel 2158716832
Fax 2158716869
Email LindonyoPCOMedu
Received 15 July 2016 | Revised 14 August 2016 | Accepted 07 September 2016
Abstract
Myocardial infarction is a form of ischemiareperfusion (IR) injury that causes cardiac contractile
dysfunction and cell death IR injury is mediated in part by decreased endothelial-derived nitric oxide (NO)
bioavailability and increased reactive oxygen species (ROS) resulting in cell death Cytokines released from
IR tissue activate G-protein coupled receptors that in turn stimulate NADPH oxidase to produce ROS Thus
administration of a NADPH oxidase peptide inhibitor Nox2ds-tat (formerly known as gp91ds-tat) may be a
rational approach to attenuate IR injury Nox2ds-tat dose-dependently inhibited (10 μM - 80 μM n=5)
phorbol 12-myristate13-acetate (n=21) induced polymorphonuclear leukocyte superoxide production up to
37 plusmn 7 (plt005 Fig 3) Similarly Nox2ds-tat dose-dependently attenuated IR induced cardiac contractile
dysfunction as evidenced by improved post-reperfused left ventricular developed pressure (LVDP) which
recovered up to 77 plusmn 7 (5 μM- 80 μM plt005 n=6-7) of initial values (pre-ischemic values) at 45 min
post-reperfusion when compared to control IR hearts (n=14) that only recovered to 46 plusmn 6 from initial
values for LVDP in isolated perfused rat hearts subjected to global I(30 min)R(45 min) (Table 1) IR
control hearts exhibited an infarct size of 46 plusmn 21 whereas IR + Nox2ds-tat hearts exhibited infarct sizes
of 30 plusmn 4 (5 μM) 15 plusmn 14 (10 μM) 23 plusmn 20 (40 microM) and 19 plusmn 16 (80 microM) (plt001 vs control IR
hearts Figure 4 Panel A-B) Regarding in vivo assessments Nox2ds-tat (41 mgkg IV) significantly
reduced blood H2O2 (14 microM) and increased endothelial-derived blood NO (127 nM) at 45 min reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 209
compared to saline controls (plt001) in rat hindlimb I(30 min)R (45 min) Moreover Nox2ds-tat (20 microM)
dose-dependently and significantly attenuated NG-L-arginine methyl ester (L-NAME) induced leukocyte
endothelial interactions up to five-fold compared to L-NAME controls (plt001) These results were
confirmed by changes seen in the histology of rat mesenteric venules (Figure 7 Panel A-B) The results
suggest that Nox2ds-tat attenuates IR-induced cardiac contractile dysfunction and infarct size by inhibiting
ROS release from NADPH oxidase
Keywords NADPH oxidase Nox2ds-tat myocardial IR hindlimb IR nitric oxide hydrogen peroxide
leukocyte superoxide release leukocyte endothelial interactions
1 Introduction
Myocardial infarction (MI) is the leading
cause of death in the United States and is
characterized by prolonged blockage of coronary
blood flow to heart tissue also known as
ischemia Reperfusion of blood to an ischemic
area is necessary to reduce the initial damage
caused by the ischemia however the process of
reperfusion itself also initiates additional injury
which may contribute up to 50 of the final
infarct size [1 2] Oxidative stress at reperfusion
is a key component of ischemiareperfusion (IR)
injury characterized by the overproduction of
oxygen-derived free radicals that can directly
damage cells These reactive oxygen species
(ROS) also diminish the bioavailability of
endothelial-derived nitric oxide (NO) resulting in
vasoconstriction and inflammation Leukocytes
are then recruited to this area of inflammation
generating a ldquorespiratory burstrdquo of superoxide
(SO) through activation of phagocytic Nox2 and
ultimately exaggerating the final infarct size [3]
Attenuating these sources of oxidative stress can
limit reperfusion-induced tissue death However
the disappointing results from non-specific anti-
oxidant agents in clinical trials suggest that it is
critical to target the sources of oxidative stress
during reperfusion rather than promote the use of
general anti-oxidants to attenuate injury after
reperfusion [2]
Nox2 is the principle isoform of NADPH
oxidase present in leukocytes and cardiovascular
tissue [4] The inflammatory process induced by
initial tissue injury up-regulates leukocyte
migration and expression of adhesion molecules
within the area of injury This augmentation of
leukocyte-endothelial interactions further
exacerbates the oxidative stress at reperfusion
leading to a decrease in NO blood concentrations
A deleterious cycle of oxidative stress and IR
injury is hereby perpetuated wherein
inflammation promotes leukocyte-endothelial
interactions and ROS production which lead to
NO quenching which in turn causes increased
inflammation and leukocyte-endothelial
interactions [3] The deleterious cycle of SO
production and NO depletion can be prevented by
identifying the source of oxidative stress and
proactively inhibiting it
There are four primary enzymatic sources
that promote oxidative stress during reperfusion
NADPH oxidases uncoupling of the
mitochondrial electron transport chain uncoupled
endothelial NO synthase (eNOS) and xanthine
oxidase activation NADPH oxidases are a unique
family of oxidases capable of exacerbating the
other three sources of ROS and reperfusion injury
(Figure 1) [4] First the assembly of cytosolic
subunits (ie Rac p67phox
p47phox
and p40phox
)
with membrane subunits (ie p22phox
and
gp91phox
) leads to the inherent production of
SOhydrogen peroxide (H2O2) upon NADPH
oxidase activation Next mitochondrial ATP-
dependent potassium channels andor
mitochondrial permeability transition pores are
augmented by the release of SOH2O2 from
activated NADPH oxidases (eg Nox2)
resulting in mitochondrial matrix alkalization
electron transport chain uncoupling and
additional SO [5] Additionally NADPH oxidase
derived SOH2O2 will cause eNOS uncoupling
and promote the formation of xanthine oxidase
via the oxidation of BH4 and xanthine
dehydrogenase respectively [6 7] Lastly
NADPH oxidase serves as a common signaling
molecule that induces the release of inflammatory
cytokines (eg TNF-α) during the first 15
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 210
minutes of reperfusion augmenting inflammation
associated injury [5-9] It is evident that NADPH
oxidase is a significant source of oxidative stress
during reperfusion both directly by producing
ROS and indirectly by propagating mediators of
IR injury
Figure 1 IR insult initially leads to cytokine mediated receptor stimulation followed by the activation of Nox2
which in turn produces SO The SO released from Nox2 induces additional ROS release from xanthine oxidase eNOS
uncoupling and dysfunctional mitochondria Collectively these sources of oxidative stress overproduce SOH2O2
species and diminish the bioavailability of blood NO This cyclical process of oxidative injury can promote further
mitochondrial dysfunction additional eNOS uncoupling and stimulate xanthine oxidase The aggregate response to
this oxidative stress at reperfusion is endothelial dysfunction leukocyte recruitment cardiomyocyte death and
increased ROS production all which lead to additional myocardial IR injury
The NADPH oxidase family contains seven
members among which Nox2 has been suggested
as an important isoform during myocardial IR
[10] First Nox2 is a ubiquitous enzyme within
several areas of the cardiovascular system such
as endothelial cells adventitial fibroblasts
myocytes and platelets and is the primary source
of SO production in Polymorphonuclear
Leukocytes (PMNs) Secondly Nox2 is located
on the cell membranes and subcellular organelles
of the aforementioned cells such as the
endoplasmic reticulum or nuclear membrane The
distribution of Nox2 allows it to affect
mitochondria and eNOS coupling in multiple
areas of the cell [11] Third Nox2 subunits can
be up-regulated by IR related mediators such as
inflammatory cytokines [12 13] Fourth Nox2
can sense the disturbance of blood flow during
periods of ischemia (ie oscillatory and eddy
current) and in response initiates vascular
inflammation which can exacerbate the initial
ischemic event [14] Lastly Nox2 deletion may
improve the cardiomyocyte survival pathway
such as the SAFE signaling pathway (ie
phosphorylation of STAT3) [15] The increase in
Nox2 activity within the cardiovascular
endothelium and leukocytes produces a
simultaneous release of SO and quenching of NO
(ie formation of peroxynitrite anion) resulting
in vascular oxidative stress endothelial
dysfunction and inflammation Studies have
shown that cardiac Nox2 knockout mice or the
delivery of Nox2 siRNA to the heart to suppress
heart Nox2 expression results in a significant
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 211
reduction of ROS production and final infarct
size in addition to improved cardiac function
following IR [16] These results are highly
suggestive that Nox2 is the primary source of SO
release in cardiac tissue
Despite previous research there are still no
published reports of any selective
pharmacological tools that address the effects of
Nox2 in IR injury and inflammation Research
involving apocynin a putative Nox2 inhibitor
was shown to exert cardioprotective effects
during reperfusion however this alkaloid of
Apocynum cannabinum can also exert direct anti-
oxidant effects independent of Nox2 inhibition
[10 17 18] Consequently our research group
chose to examine Nox2ds-tat (formerly known as
gp91ds-tat) a more selective Nox2 peptide
inhibitor to better understand the specific role of
Nox2 in propagating oxidative stress during IR
injury The tat portion of this peptide contains a
9-aa sequence (ie [H]- R-K-K-R-R-Q-R-R-R )
which facilitates Nox2ds-tat delivery into the cell
The docking sequence (ie C-S-T-R-R-R-Q-Q-
L-NH2) specifically inhibits the assembly of
cytosolic subunit p47phox
with the membrane
subunit gp91 and subsequently inhibits Nox2
assembly and activation (Figure 2) [19 20 21]
Moreover in recent studies Nox2ds-tat has been
shown to suppress balloon angioplasty induced
SO production in carotid artery ischemia
suggesting that the effects of Nox2ds-tat could be
extended to cardiomyocyte IR injury [21 22] It
is evident that Nox2ds-tat is a promising NADPH
oxidase inhibitor selectively targeting Nox2
which prevents the deleterious cycle of SO
production and NO depletion characteristic of
reperfusion-induced myocardial injury
Figure 2 Schematic of Nox2ds-tat mechanism of action whereby the blue line shows inhibition of cytosolic subunit
p47phox on the gp91 Nox2 membrane component This disallows for NADPH oxidase to assemble and the inherent
production of ROS with this event as depicted with the blue cross out Adapted from Wilkinson et al [40]
In this study we first determined the dose-
dependent effects of Nox2ds-tat on leukocyte SO
production Next the effects of this peptide were
evaluated in myocardial and hindlimb IR by
post-reperfused cardiac functioninfarct size and
NOH2O2 respectively We also studied the
effects of the peptide on NG-L-arginine methyl
ester (L-NAME) induced leukocyte-endothelial
interactions in rat mesenteric venules by intravital
microscopy We predict that Nox2ds-tat will
attenuate ROS in phorbol 12-myristate 13-acetate
(PMA) induced leukocytes and hindlimb IR will
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 212
decrease infarct size in myocardial IR and will
attenuate leukocyte-endothelial interactions in rat
mesenteric venules Moreover we predict that the
reduction in infarct size will parallel an
improvement in post reperfusion cardiac function
2 Methods
21 Animals
All animal procedures were approved by the
Institutional Animal Care and Use Committee at
PCOM for care and use of animals Male Sprague
Dawley (SD) rats (275-325 g Charles River
Springfield MA) were used for ex vivo and in
vivo experiments and 350-400 g male SD rats
were used for SO release assays listed below
22 Measurement of SO Release from Rat
PMNs
PMNs were isolated from male SD rats and
SO release was measured spectrophotometrically
(model 260 Gilford Nova Biotech El Cajon CA)
by the reduction of ferricytochrome c [23] The
PMNs (5x106) were re-suspended in 450 μl PBS
and incubated with ferricytochrome c (100 microM
Sigma Chemical) in a total volume of 900 μl PBS
in the presence or absence of varying
concentrations of Nox2ds-tat peptide inhibitor
([H]- R-K-K-R-R-Q-R-R-R-C-S-T-R-R-R-Q-Q-
L-NH2 MW=2452 gmol Genemed Synthesis
San Antoino TX) 10 M 40 M or 80 μM
respectively for 15 min at 37degC in
spectrophotometric cells The PMNs were
stimulated with 100 nM PMA (MW= 6168
gmol Calbiochem) in a final reaction volume of
10 ml Absorbance at 550 nm was measured
every 30 seconds for up to 360 seconds and the
change in absorbance (SO release) from PMNs
was determined relative to time 0 The dose-
response curve of Nox2ds-tat on PMN SO release
was used to determine a dose range that may be
effective in myocardial IR experiments
23 Isolated Rat Heart Preparation
Male SD rats were anesthetized
intraperitoneally (IP) with 60 mgkg pentobarbital
sodium and anticoagulated with 1000 U of
sodium heparin IP Plasma was prepared from
blood isolated from the abdominal aorta of the
same rat from which the heart was isolated from
for each cardiac perfusion experiment The
plasma was used as the vehicle for infusion of
Nox2ds-tat The hearts were rapidly excised and
perfused via the Langendorff heart preparation
[24] The aorta was cannulated and retrograde
perfused with modified Krebsrsquo buffer containing
170 mM dextrose 1200 mM NaCl 250 mM
NaHCO3 25 mM CaCl2 05 mM EDTA 59
mM KCl and 12 mM MgCl2 The modified
Krebsrsquo buffer was maintained at 37oC 80 mmHg
constant pressure aerated with 95 O2-5 CO2
and equilibrated pH between 73-74 Hearts were
immersed in an H2O jacket reservoir containing
160 ml of modified Krebsrsquo buffer at 37oC to
provide the preload The preload was established
by the volume of modified Krebsrsquo buffer that
entered the left ventricle The pressure transducer
catheter was inserted into the left ventricle of the
heart through the mitral valve Hearts were
subjected to 15 min Krebsrsquo buffer perfusion for
stabilization in order to record an initial baseline
then 30 min of global ischemia by stopping
perfusion followed by a 45 min reperfusion
period A volume of 5 ml of plasma (control) or
plasma containing Nox2ds-tat (5 μM 10 μM 40
μM or 80 μM) was injected during the first 5 min
of reperfusion by a side arm line proximal to the
heart inflow cannula at a rate of 1 mlmin All
cardiac function including coronary flow left
ventricular developed pressure ([LVDP] which is
the left ventricular end-systolic pressure [LVESP]
minus left ventricular end-diastolic pressure
[LVEDP]) maximal and minimal rate of LVDP
(+dPdtmax and ndashdPdtmin) and heart rate readings
(BPM) were taken every 5 min from initial
baseline and then throughout reperfusion
Coronary flow (mlmin) was recorded using an
inline flow meter (T106 Transonic Systems Inc
Ithaca NY) and LVDP LVESP LVEDP
+dPdtmax ndashdPdtmin and BPM readings were
recorded using a pressure transducer (SPR-524
Millar Instruments Inc Houston TX) Data was
recorded using a Powerlab Station acquisition
system (ADInstruments Grand Junction CO) At
the end of the experiment the left ventricle was
isolated and cross sectioned into 2mm pieces
from apex to base and was subjected to 1
triphenyltetrazolium chloride (TTC) staining for
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 213
15 min at 37oC to detect infarct size as previously
described [24]
24 Hindlimb IR in vivo procedure
We measured blood H2O2NO release in real-
time from sensors placed in the femoral veins of
male SD rats anesthetized with sodium
pentobarbital (60 mgkg IP for induction and 30
mgkg IP for maintenance) Thereafter one limb
was subjected to IR while the other limb was
used as a non-ischemic sham control in the same
rat The calibrated H2O2 or NO microsensors
(100 microm World Precision Instruments [WPI] Inc
Sarasota FL) were connected to a free radical
analyzer (Apollo 4000 WPI Inc) and inserted
into a 24 gauge catheter placed inside each
femoral vein as previously described by Chen et
al [25] and reviewed by Young et al [26]
Ischemia in one hindlimb was induced by
clamping the femoral arteryvein for 30 min
followed by 45 min of reperfusion Nox2ds-tat
(41 mgkg prepared in saline ~20 μM in blood)
or saline (non-drug control group) was
administered at the beginning of reperfusion as a
bolus via the jugular vein We continuously
recorded and collected the H2O2 or NO release
data at 5 min intervals during a 15 min baseline
30 min ischemia and 45 min reperfusion period
The changes in H2O2 or NO release (in pA)
during IR are expressed as relative change to
initial readings Thereafter the values were
converted to the concentration of H2O2 (μM) or
NO (nM) after correction to the respective
calibration curves The values recorded from the
femoral vein of the IR limb were subtracted from
the values of the sham limb to yield a net
difference and were recorded every 5 min
continuously on time course graphs [25]
25 Intravital Microscopy
Male SD rats were anesthetized with 60
mgkg pentobarbital sodium IP and maintained
with 30 mgkg pentobarbital IP The left carotid
artery was isolated and a PE-50 polyethylene
catheter was inserted in the left carotid artery for
monitoring of the mean arterial blood pressure
(MABP) via BP-1 Pressure Monitor (WPI Inc
Sarasota FL) After abdominal laparotomy a
loop of ileal mesentery was exteriorized and
placed in a temperature controlled Plexiglas
chamber (37 degC) for adequate superfusion of the
test solutions [27] The mesentery was placed
over a Plexiglas pedestal in the observation
chamber for visualization under a Nikon Eclipse
microscope (Nikon Co Japan) The
microcirculation was recorded with Image Pro
MDA (Media Cybernetics Bethesda MD) and
leukocyte-endothelial interactions were analyzed
offline
The rats were allowed to stabilize for 30 min
with superfusion of Krebsrsquo buffer after surgery
After stabilization a non-branched postcapillary
venule was chosen for observation An initial
recording was made to establish basal values for
leukocyte rolling adherence and transmigration
The mesentery then was superfused with the
experimental test solutions for 120 min Test
solutions included Krebsrsquo buffer alone 50 M L-
NAME alone and 50 M L-NAME in the
presence of Nox2ds-tat (5 M or 20 M) All
drugs were dissolved into the Krebsrsquo buffer The
solution was aerated with 95 N2-5 CO2 and
equilibrated at a pH of 73 to 74 at 37 degC Two
min video recordings were made at baseline 30
min 60 min 90 min and 120 min after
superfusion of the test solutions for quantification
of leukocyte rolling adherence and
transmigration [24 27]
26 Hematoxylin and Eosin (HampE) Staining After the experiment the loop of the ileal
mesentery that was superfused during the
experiment was removed and quickly stored in 4
paraformaldehyde for histological analysis of
leukocyte adherence and transmigration by HampE
staining Three representative sections of the ileal
mesentery from Krebsrsquo buffer 50 microM L-NAME
and 50 microM L-NAME in the presence of Nox2ds-
tat (5 M or 20 microM) were selected for
histological analysis Tissue samples were
selected from experiments representative of the
group mean of intravital microscopy (Table 2)
The tissue was embedded in paraffin and
sectioned into 45 microm serial sections and placed
onto glass slides Sections were deparaffinized
and rehydrated then stained with HampE [27]
Under light microscopy leukocyte adherence and
transmigration was counted in areas containing
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 214
venulesarterioles within the serosa and the
mesentery and expressed as adhered and
transmigrated leukocytesmm2 in tissue
27 Statistical Analysis
All data in the text and figures are presented
as means plusmn standard error of the mean (SEM)
The data was analyzed by analysis of variance
(ANOVA) using post hoc analysis with the
Student-Newman-Keuls test for all experiments
except for hindlimb IR in which a studentrsquos t-test
was used A p value of lt005 was considered to
be statistically significant
3 Results
31 PMN SO release
PMA is a broad-spectrum activator of protein
kinase C (PKC) which is essential in promoting
NADPH oxidase assembly at the PMN
membrane to generate SO release [28]We found
that Nox2ds-tat exhibited a dose-dependent
reduction in PMA-stimulated PMN SO release
(Figure 3) Doses of Nox2ds-tat (10 μM and 40
μM) did not significantly decrease PMN SO
release whereas a dose of 80 μM significantly
inhibited PMA-induced SO release by 37 plusmn 7
(plt005 compared to PMA alone) The data
generated from this assay was used to test a
similar dose range of Nox2ds-tat in isolated rat
heart IR experiments
Figure 3 PMA (100 nM) induced SO release in PMNs peak response read at 360s post stimulation Nox2ds-tat dose
dependently inhibited SO release from PMNs by 37 plusmn 7 (80 microM n=4) when compared to PMNs treated with PMA
alone (plt005 vs PMA control)
32 Cardiac function and infarct size
Nox2ds-tat given at reperfusion dose-
dependently attenuated IR induced cardiac
contractile dysfunction and recovered post-
reperfused LVDP to 47 plusmn 7 (5 μM n=7) 69 plusmn
13 (10 μM n=6 plt005) 68 plusmn 7 (40 μM
n=6 plt005) and 77 plusmn 7 (80 μM n=6 plt005)
of initial LVDP at 45 min post-reperfusion when
compared to control IR hearts that only
recovered to 46 plusmn 6 (n=14) (Table 1) Initial
baseline values for cardiac function parameters
did not differ significantly among groups
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 215
Table 1 Cardiac function parameters among different experimental groups
Cardiac Parameters Control IR
(n=14)
IR +
Nox2ds-tat 5
μM
(n=7)
IR +
Nox2ds-tat
10 μM
(n=6)
IR +
Nox2ds-tat
40 μM
(n=6)
IR +
Nox2ds-tat
80 μM
(n=6)
Initial LVDP (mmHg) 87plusmn35 91plusmn31 89plusmn34 92plusmn39 88plusmn33
Final LVDP (mmHg) 40plusmn50 43plusmn66 61plusmn121 63plusmn62 68plusmn62
Initial LVESP (mmHg) 99plusmn38 101plusmn39 105plusmn48 103plusmn46 101plusmn37
Final LVESP (mmHg) 100plusmn53 108plusmn57 107plusmn119 110plusmn75 124plusmn97
Initial LVEDP (mmHg) 11plusmn10 94plusmn13 16plusmn20 11plusmn13 13plusmn07
Final LVEDP(mmHg) 61plusmn51 64plusmn42 46plusmn61 47plusmn75
56plusmn56
Initial +dPdtmax (mmHgsec) 2259plusmn71 2335plusmn61 2333plusmn66 2346plusmn77 2277plusmn79
Final +dPdtmax (mmHgsec) 894plusmn105 832plusmn124 1285plusmn232 1258plusmn120
1413plusmn100
Initial -dPdtmin (mmHgsec) -1510plusmn67 -1616plusmn98 -1607plusmn84 -1550plusmn124 -1524plusmn106
Final -dPdtmin (mmHgsec) -704plusmn65 -660plusmn136 -879plusmn187 -939plusmn108 -1116plusmn146
Initial coronary flow
(mlmin) 162plusmn12 144plusmn064 181plusmn14 180plusmn25 152plusmn10
Final coronary flow (mlmin) 79plusmn064 79plusmn070 92plusmn15 93plusmn17 95plusmn15
Initial Heart Rate (BPM) 271plusmn128 277plusmn123 280plusmn78 272plusmn88 265plusmn32
Final Heart Rate (BPM) 246plusmn188 234plusmn199 230plusmn105 249plusmn136 237plusmn119
plt005 vs control IR final cardiac function parameters plt005
plt001 vs Nox2ds-tat 5μM
Similarly Nox2ds-tat reduced infarct
sizearea-at-risk when compared to the IR
control group (Figure 4 Panel A-B) The cross
section for the IR control group shows the
majority of the infarct in the mid-wall area
whereas the Nox2ds-tat treated IR hearts showed
less infarct area (Figure 4 Panel A) Figure 4
Panel B shows the ratio of this tissue at risk
When compared to the IR control group (46 plusmn
2) there was a significant decrease in total
infarct size in hearts treated with Nox2ds-tat 30 plusmn
4 (5 μM) 15 plusmn 14 (10 μM) 23 plusmn 2 (40
microM) and 19 plusmn 16 (80 microM)(plt001 for all
groups) Moreover the 10 μM and 80 μM doses
showed significantly less infarct size compared to
the 5 μM dose (plt001 and plt005 respectively)
suggesting a dose-dependent effect of Nox2ds-tat
on infarct size (Figure 4 Panel A-B)
Other cardiac function parameters are
illustrated in Table 1 Of interest Nox2ds-tat
dose-dependently recovered post-reperfused final
+dPdtmax by 36 plusmn 53 (5 μM) 55 plusmn10 (10
μM) 54 plusmn 4 (40 microM plt005) 62 plusmn 4 (80 microM
plt005) at 45 min reperfusion compared to
control IR hearts that only recovered to 40 plusmn 5
Sham hearts (n=6) were not subjected to IR and
maintained cardiac function parameters and flow
by gt90 and had lt5 infarct sizearea-at- risk
throughout the experimental time-course (data
not shown)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 216
Figure 4 Panel A Representative images of TTC staining and percentage of left ventricular heart tissue at risk among
different experimental groups Viable tissue stains red infarcted tissue did not retain TTC staining Nox2ds-tat dose
dependently attenuated infarct size Panel B Ratio of left ventricular heart tissue at risk for myocardial dysfunction
out of total tissue weight as determined by TTC staining of representative heart sections When compared to the IR
control group which had an infarct size of 46plusmn2 there was a significant decrease in total infarct size in heart sections
treated with Nox2ds-tat Nox2ds-tat dose-dependently attenuated infarct sizes to 30 plusmn 4 (5 μM n=7) 15 plusmn 14 (10
μM n=6) 23 plusmn 2 (40 microM n=6) and 19 plusmn 16 (80 microM n=6) (plt001 vs control tissue n=14) There was also a
significant decrease in infarct size observed in 10 μM (plt001) and 80 microM (plt005) compared to 5 μM Nox2ds-tat
(plt001 vs control IR plt005 vs Nox2ds-tat 5 microM plt001 vs Nox2ds-tat 5microM)
33 Hindlimb IR in vivo
331 Nox2ds-tat reduced blood H2O2 levels
The blood H2O2 values in the IR hindlimb of
saline controls significantly increased to 17 plusmn 03
microM (15 min post-reperfusion) above sham values
Thereafter a relative difference of 12-14 μM
between the IR and sham limbs was maintained
throughout the remainder of reperfusion (Figure 5
Panel A) By contrast Nox2ds-tat (41 mgkg
~20 μM in blood) treated animals in the IR
hindlimb did not significantly differ from the
sham hindlimb throughout reperfusion (Figure 5
Panel B) The relative difference in blood H2O2
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant reduction in the IR induced effect on
blood H2O2 by 143 plusmn 02 μM at the end of
reperfusion (Figure 5 Panel C)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 217
Figure 5 Panel A Relative difference in measured blood H2O2 between IR and sham femoral veins in saline controls
The saline IR vein showed a significant increase in blood H2O2 by 17 plusmn 03 microM (5 min) relative to the sham femoral
vein This increase above sham femoral vein values was maintained throughout reperfusion stabilizing at 14 plusmn 02 microM
(45 min) (plt005 plt001 vs sham IR femoral vein) Panel B Relative difference in measured blood H2O2
between IR and sham femoral veins in Nox2ds-tat (41 mgkg ~20 microM) treated rats The IR femoral vein treated with
Nox2ds-tat showed a transient increase in H2O2 when compared to the sham femoral vein However this difference
between the two limbs decreased throughout the remainder of the experiment ending at ~0 microM The measured values
in the two groups were not statistically different at any point in the experiment Panel C Relative difference in
measured blood H2O2 betweenNox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline
values) Nox2ds-tat infusion at reperfusion significantly decreased the blood H2O2 concentration when compared to
rats infused with saline At the end of the 45 min reperfusion period there was a significant reduction of 14 microM
between blood H2O2 measured in Nox2ds-tat 20 microM treated rats relative to saline control (plt005 plt001 vs
saline treated femoral veins)
B
A
C
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 218
A
B
C
Figure 6 Panel A Relative difference in measured blood NO between IR and sham femoral veins in saline controls
Overall the IR femoral vein showed a significant decrease in NO concentration stabilizing at a difference of 90 plusmn 21
nM when compared to the sham femoral vein at the end of the 45 min reperfusion period (plt005 plt001 vs sham
IR femoral vein) Panel B Relative difference in measured blood NO between IR and sham femoral veins in Nox2ds-
tat (41 mgkg ~20 microM) treated rats In Nox2ds-tat treated animals the IR femoral vein resulted in 37 plusmn 12 nM higher
blood NO concentration than the sham limb at the end of the 45 min reperfusion period however this difference was
not statistically significant (plt005 plt001 vs sham IR femoral vein) Panel C Relative difference in measured
blood NO in Nox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline values) Nox2ds-tat
given at reperfusion significantly increased the total blood NO concentration when compared to saline controls At the
end of the 45 min reperfusion period there was a significant increase of 127 nM in blood NO in Nox2ds-tat treated
animals relative to saline controls (plt005 plt001 vs saline treated femoral veins)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 219
332 Nox2ds-tat prevented the decrease in
blood NO levels
In saline controls blood NO levels steadily
decreased in the IR limb throughout the
reperfusion period leading to significantly lower
blood NO bioavailability (90 nM below sham) as
early as 20 min into the reperfusion period
(Figure 6 Panel A) By contrast blood NO in the
IR hindlimb of Nox2ds-tat (41 mgkg ~20 μM
in blood) treated animals did not significantly
differ from sham hindlimb values during the
reperfusion period While a linear trend close to
sham values was observed the Nox2ds-tat treated
grouprsquos blood NO levels did rise to 37 nM above
sham values at the end of reperfusion (Figure 6
Panel B) The relative difference in blood NO
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant increase in the IR induced effect on
NO release by 127 nM at the end of reperfusion
(Figure 6 Panel C)
34 L-NAME induced Leukocyte endothelial
interactions
341 Intravital Microscopy
There was no significant difference among
initial baseline values in leukocyte rolling
adherence and transmigration among all study
groups In all parameters the Krebsrsquo buffer group
did not significantly change from baseline L-
NAME (50 microM) significantly increased leukocyte
rolling adherence and transmigration when
compared to Krebsrsquo at T=120 min by 82 plusmn 11
(plt001) 22 plusmn 5 (plt001) and 21 plusmn 4 (plt001)
respectively (Table 2) Nox2ds-tat (5 microM) did not
significantly attenuate L-NAME induced
leukocyte-endothelial interactions except for final
transmigration (plt005) By contrast Nox2ds-tat
(20 microM) significantly decreased the L-NAME
induced effect by 3-5 fold on all parameters
(plt001) (Table 2)
Table 2 The comparision of initial and final (120 min of reperfusion) leukocyte rolling adherence and transmigration
between Krebsrsquo L-NAME and L-NAME + Nox2ds-tat (5 μM 20 μM) groups in rat mesenteric venules
Intravital microscopy Parameters
Krebsrsquo
(n=6)
L-NAME
(n=5)
L-NAME +
Nox2ds-tat 5 μM
(n=4)
L-NAME +
Nox2ds-tat 20 μM
(n=6)
Initial Rolling (numbermin) 8plusmn3 11plusmn3 13plusmn3 8plusmn2
Final Rolling (numbermin) 16plusmn5 82plusmn11
72plusmn13 16plusmn6++
Initial Adherence (number100um) 2plusmn2 1plusmn0 3plusmn0 1plusmn1
Final Adherence (number100um) 4plusmn2 22plusmn5 17plusmn1 8plusmn4
Initial Transmigration (number) 1plusmn0 1plusmn0 1plusmn0 1plusmn0
Final Transmigration (number) 1plusmn0 21plusmn4 9plusmn1
4plusmn2
Plt005 Plt001 compared to Krebsrsquo groupPlt005
Plt001 compared to L-NAME
++Plt001 compared to L-
NAME + Nox2ds-tat 5 μM
342 HampE Staining
The Krebsrsquocontrol group exhibited only 73 plusmn
33 and 62 plusmn 30 leukocytesmm2 of mesenteric
tissue These values represent basal numbers of
leukocyte adherence and transmigration in this
tissue bed respectively L-NAME treatment
exhibited a marked increase (~4-fold) in
leukocyte vascular adherence and transmigration
compared to Krebsrsquo buffer (299 plusmn 23 and 271 plusmn
26 leukocytesmm2 of tissue respectively plt001
for both values) Nox2ds-tat dose-dependently
attenuated the L-NAME induced effect on
leukocyte adherence (199 plusmn 13 for 5 microM at
plt005 and 83 plusmn 9 for 20 microM plt001) and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
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101074jbcM209793200
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(2011) Central TNF inhibition results in
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14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
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101161CIRCULATIONAHA113002714
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M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
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107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
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infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
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1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
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101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
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triumphs and pitfalls Antioxidants amp Redox
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101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
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10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
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phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
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249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
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105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
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(2005) Go 6983 a fast acting protein kinase
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101111j1527-34662005tb00170x
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Mechanisms for the role of
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and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
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R M (1994) Role of the membrane cortex
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Cytoskeletal regulation of membrane
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33 Manceur A Wu A amp Audet J (2007)
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35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
Immunology 33(5) 1328-1333 DOI
101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
inhibition attenuates myocardial infarction
induced heart failure and is associated with a
reduction of fibrosis and pro‐inflammatory
responses Journal of Cellular and
Molecular Medicine 15(8) 1769-1777 DOI
101111j1582-4934201001174x
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 209
compared to saline controls (plt001) in rat hindlimb I(30 min)R (45 min) Moreover Nox2ds-tat (20 microM)
dose-dependently and significantly attenuated NG-L-arginine methyl ester (L-NAME) induced leukocyte
endothelial interactions up to five-fold compared to L-NAME controls (plt001) These results were
confirmed by changes seen in the histology of rat mesenteric venules (Figure 7 Panel A-B) The results
suggest that Nox2ds-tat attenuates IR-induced cardiac contractile dysfunction and infarct size by inhibiting
ROS release from NADPH oxidase
Keywords NADPH oxidase Nox2ds-tat myocardial IR hindlimb IR nitric oxide hydrogen peroxide
leukocyte superoxide release leukocyte endothelial interactions
1 Introduction
Myocardial infarction (MI) is the leading
cause of death in the United States and is
characterized by prolonged blockage of coronary
blood flow to heart tissue also known as
ischemia Reperfusion of blood to an ischemic
area is necessary to reduce the initial damage
caused by the ischemia however the process of
reperfusion itself also initiates additional injury
which may contribute up to 50 of the final
infarct size [1 2] Oxidative stress at reperfusion
is a key component of ischemiareperfusion (IR)
injury characterized by the overproduction of
oxygen-derived free radicals that can directly
damage cells These reactive oxygen species
(ROS) also diminish the bioavailability of
endothelial-derived nitric oxide (NO) resulting in
vasoconstriction and inflammation Leukocytes
are then recruited to this area of inflammation
generating a ldquorespiratory burstrdquo of superoxide
(SO) through activation of phagocytic Nox2 and
ultimately exaggerating the final infarct size [3]
Attenuating these sources of oxidative stress can
limit reperfusion-induced tissue death However
the disappointing results from non-specific anti-
oxidant agents in clinical trials suggest that it is
critical to target the sources of oxidative stress
during reperfusion rather than promote the use of
general anti-oxidants to attenuate injury after
reperfusion [2]
Nox2 is the principle isoform of NADPH
oxidase present in leukocytes and cardiovascular
tissue [4] The inflammatory process induced by
initial tissue injury up-regulates leukocyte
migration and expression of adhesion molecules
within the area of injury This augmentation of
leukocyte-endothelial interactions further
exacerbates the oxidative stress at reperfusion
leading to a decrease in NO blood concentrations
A deleterious cycle of oxidative stress and IR
injury is hereby perpetuated wherein
inflammation promotes leukocyte-endothelial
interactions and ROS production which lead to
NO quenching which in turn causes increased
inflammation and leukocyte-endothelial
interactions [3] The deleterious cycle of SO
production and NO depletion can be prevented by
identifying the source of oxidative stress and
proactively inhibiting it
There are four primary enzymatic sources
that promote oxidative stress during reperfusion
NADPH oxidases uncoupling of the
mitochondrial electron transport chain uncoupled
endothelial NO synthase (eNOS) and xanthine
oxidase activation NADPH oxidases are a unique
family of oxidases capable of exacerbating the
other three sources of ROS and reperfusion injury
(Figure 1) [4] First the assembly of cytosolic
subunits (ie Rac p67phox
p47phox
and p40phox
)
with membrane subunits (ie p22phox
and
gp91phox
) leads to the inherent production of
SOhydrogen peroxide (H2O2) upon NADPH
oxidase activation Next mitochondrial ATP-
dependent potassium channels andor
mitochondrial permeability transition pores are
augmented by the release of SOH2O2 from
activated NADPH oxidases (eg Nox2)
resulting in mitochondrial matrix alkalization
electron transport chain uncoupling and
additional SO [5] Additionally NADPH oxidase
derived SOH2O2 will cause eNOS uncoupling
and promote the formation of xanthine oxidase
via the oxidation of BH4 and xanthine
dehydrogenase respectively [6 7] Lastly
NADPH oxidase serves as a common signaling
molecule that induces the release of inflammatory
cytokines (eg TNF-α) during the first 15
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 210
minutes of reperfusion augmenting inflammation
associated injury [5-9] It is evident that NADPH
oxidase is a significant source of oxidative stress
during reperfusion both directly by producing
ROS and indirectly by propagating mediators of
IR injury
Figure 1 IR insult initially leads to cytokine mediated receptor stimulation followed by the activation of Nox2
which in turn produces SO The SO released from Nox2 induces additional ROS release from xanthine oxidase eNOS
uncoupling and dysfunctional mitochondria Collectively these sources of oxidative stress overproduce SOH2O2
species and diminish the bioavailability of blood NO This cyclical process of oxidative injury can promote further
mitochondrial dysfunction additional eNOS uncoupling and stimulate xanthine oxidase The aggregate response to
this oxidative stress at reperfusion is endothelial dysfunction leukocyte recruitment cardiomyocyte death and
increased ROS production all which lead to additional myocardial IR injury
The NADPH oxidase family contains seven
members among which Nox2 has been suggested
as an important isoform during myocardial IR
[10] First Nox2 is a ubiquitous enzyme within
several areas of the cardiovascular system such
as endothelial cells adventitial fibroblasts
myocytes and platelets and is the primary source
of SO production in Polymorphonuclear
Leukocytes (PMNs) Secondly Nox2 is located
on the cell membranes and subcellular organelles
of the aforementioned cells such as the
endoplasmic reticulum or nuclear membrane The
distribution of Nox2 allows it to affect
mitochondria and eNOS coupling in multiple
areas of the cell [11] Third Nox2 subunits can
be up-regulated by IR related mediators such as
inflammatory cytokines [12 13] Fourth Nox2
can sense the disturbance of blood flow during
periods of ischemia (ie oscillatory and eddy
current) and in response initiates vascular
inflammation which can exacerbate the initial
ischemic event [14] Lastly Nox2 deletion may
improve the cardiomyocyte survival pathway
such as the SAFE signaling pathway (ie
phosphorylation of STAT3) [15] The increase in
Nox2 activity within the cardiovascular
endothelium and leukocytes produces a
simultaneous release of SO and quenching of NO
(ie formation of peroxynitrite anion) resulting
in vascular oxidative stress endothelial
dysfunction and inflammation Studies have
shown that cardiac Nox2 knockout mice or the
delivery of Nox2 siRNA to the heart to suppress
heart Nox2 expression results in a significant
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 211
reduction of ROS production and final infarct
size in addition to improved cardiac function
following IR [16] These results are highly
suggestive that Nox2 is the primary source of SO
release in cardiac tissue
Despite previous research there are still no
published reports of any selective
pharmacological tools that address the effects of
Nox2 in IR injury and inflammation Research
involving apocynin a putative Nox2 inhibitor
was shown to exert cardioprotective effects
during reperfusion however this alkaloid of
Apocynum cannabinum can also exert direct anti-
oxidant effects independent of Nox2 inhibition
[10 17 18] Consequently our research group
chose to examine Nox2ds-tat (formerly known as
gp91ds-tat) a more selective Nox2 peptide
inhibitor to better understand the specific role of
Nox2 in propagating oxidative stress during IR
injury The tat portion of this peptide contains a
9-aa sequence (ie [H]- R-K-K-R-R-Q-R-R-R )
which facilitates Nox2ds-tat delivery into the cell
The docking sequence (ie C-S-T-R-R-R-Q-Q-
L-NH2) specifically inhibits the assembly of
cytosolic subunit p47phox
with the membrane
subunit gp91 and subsequently inhibits Nox2
assembly and activation (Figure 2) [19 20 21]
Moreover in recent studies Nox2ds-tat has been
shown to suppress balloon angioplasty induced
SO production in carotid artery ischemia
suggesting that the effects of Nox2ds-tat could be
extended to cardiomyocyte IR injury [21 22] It
is evident that Nox2ds-tat is a promising NADPH
oxidase inhibitor selectively targeting Nox2
which prevents the deleterious cycle of SO
production and NO depletion characteristic of
reperfusion-induced myocardial injury
Figure 2 Schematic of Nox2ds-tat mechanism of action whereby the blue line shows inhibition of cytosolic subunit
p47phox on the gp91 Nox2 membrane component This disallows for NADPH oxidase to assemble and the inherent
production of ROS with this event as depicted with the blue cross out Adapted from Wilkinson et al [40]
In this study we first determined the dose-
dependent effects of Nox2ds-tat on leukocyte SO
production Next the effects of this peptide were
evaluated in myocardial and hindlimb IR by
post-reperfused cardiac functioninfarct size and
NOH2O2 respectively We also studied the
effects of the peptide on NG-L-arginine methyl
ester (L-NAME) induced leukocyte-endothelial
interactions in rat mesenteric venules by intravital
microscopy We predict that Nox2ds-tat will
attenuate ROS in phorbol 12-myristate 13-acetate
(PMA) induced leukocytes and hindlimb IR will
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 212
decrease infarct size in myocardial IR and will
attenuate leukocyte-endothelial interactions in rat
mesenteric venules Moreover we predict that the
reduction in infarct size will parallel an
improvement in post reperfusion cardiac function
2 Methods
21 Animals
All animal procedures were approved by the
Institutional Animal Care and Use Committee at
PCOM for care and use of animals Male Sprague
Dawley (SD) rats (275-325 g Charles River
Springfield MA) were used for ex vivo and in
vivo experiments and 350-400 g male SD rats
were used for SO release assays listed below
22 Measurement of SO Release from Rat
PMNs
PMNs were isolated from male SD rats and
SO release was measured spectrophotometrically
(model 260 Gilford Nova Biotech El Cajon CA)
by the reduction of ferricytochrome c [23] The
PMNs (5x106) were re-suspended in 450 μl PBS
and incubated with ferricytochrome c (100 microM
Sigma Chemical) in a total volume of 900 μl PBS
in the presence or absence of varying
concentrations of Nox2ds-tat peptide inhibitor
([H]- R-K-K-R-R-Q-R-R-R-C-S-T-R-R-R-Q-Q-
L-NH2 MW=2452 gmol Genemed Synthesis
San Antoino TX) 10 M 40 M or 80 μM
respectively for 15 min at 37degC in
spectrophotometric cells The PMNs were
stimulated with 100 nM PMA (MW= 6168
gmol Calbiochem) in a final reaction volume of
10 ml Absorbance at 550 nm was measured
every 30 seconds for up to 360 seconds and the
change in absorbance (SO release) from PMNs
was determined relative to time 0 The dose-
response curve of Nox2ds-tat on PMN SO release
was used to determine a dose range that may be
effective in myocardial IR experiments
23 Isolated Rat Heart Preparation
Male SD rats were anesthetized
intraperitoneally (IP) with 60 mgkg pentobarbital
sodium and anticoagulated with 1000 U of
sodium heparin IP Plasma was prepared from
blood isolated from the abdominal aorta of the
same rat from which the heart was isolated from
for each cardiac perfusion experiment The
plasma was used as the vehicle for infusion of
Nox2ds-tat The hearts were rapidly excised and
perfused via the Langendorff heart preparation
[24] The aorta was cannulated and retrograde
perfused with modified Krebsrsquo buffer containing
170 mM dextrose 1200 mM NaCl 250 mM
NaHCO3 25 mM CaCl2 05 mM EDTA 59
mM KCl and 12 mM MgCl2 The modified
Krebsrsquo buffer was maintained at 37oC 80 mmHg
constant pressure aerated with 95 O2-5 CO2
and equilibrated pH between 73-74 Hearts were
immersed in an H2O jacket reservoir containing
160 ml of modified Krebsrsquo buffer at 37oC to
provide the preload The preload was established
by the volume of modified Krebsrsquo buffer that
entered the left ventricle The pressure transducer
catheter was inserted into the left ventricle of the
heart through the mitral valve Hearts were
subjected to 15 min Krebsrsquo buffer perfusion for
stabilization in order to record an initial baseline
then 30 min of global ischemia by stopping
perfusion followed by a 45 min reperfusion
period A volume of 5 ml of plasma (control) or
plasma containing Nox2ds-tat (5 μM 10 μM 40
μM or 80 μM) was injected during the first 5 min
of reperfusion by a side arm line proximal to the
heart inflow cannula at a rate of 1 mlmin All
cardiac function including coronary flow left
ventricular developed pressure ([LVDP] which is
the left ventricular end-systolic pressure [LVESP]
minus left ventricular end-diastolic pressure
[LVEDP]) maximal and minimal rate of LVDP
(+dPdtmax and ndashdPdtmin) and heart rate readings
(BPM) were taken every 5 min from initial
baseline and then throughout reperfusion
Coronary flow (mlmin) was recorded using an
inline flow meter (T106 Transonic Systems Inc
Ithaca NY) and LVDP LVESP LVEDP
+dPdtmax ndashdPdtmin and BPM readings were
recorded using a pressure transducer (SPR-524
Millar Instruments Inc Houston TX) Data was
recorded using a Powerlab Station acquisition
system (ADInstruments Grand Junction CO) At
the end of the experiment the left ventricle was
isolated and cross sectioned into 2mm pieces
from apex to base and was subjected to 1
triphenyltetrazolium chloride (TTC) staining for
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 213
15 min at 37oC to detect infarct size as previously
described [24]
24 Hindlimb IR in vivo procedure
We measured blood H2O2NO release in real-
time from sensors placed in the femoral veins of
male SD rats anesthetized with sodium
pentobarbital (60 mgkg IP for induction and 30
mgkg IP for maintenance) Thereafter one limb
was subjected to IR while the other limb was
used as a non-ischemic sham control in the same
rat The calibrated H2O2 or NO microsensors
(100 microm World Precision Instruments [WPI] Inc
Sarasota FL) were connected to a free radical
analyzer (Apollo 4000 WPI Inc) and inserted
into a 24 gauge catheter placed inside each
femoral vein as previously described by Chen et
al [25] and reviewed by Young et al [26]
Ischemia in one hindlimb was induced by
clamping the femoral arteryvein for 30 min
followed by 45 min of reperfusion Nox2ds-tat
(41 mgkg prepared in saline ~20 μM in blood)
or saline (non-drug control group) was
administered at the beginning of reperfusion as a
bolus via the jugular vein We continuously
recorded and collected the H2O2 or NO release
data at 5 min intervals during a 15 min baseline
30 min ischemia and 45 min reperfusion period
The changes in H2O2 or NO release (in pA)
during IR are expressed as relative change to
initial readings Thereafter the values were
converted to the concentration of H2O2 (μM) or
NO (nM) after correction to the respective
calibration curves The values recorded from the
femoral vein of the IR limb were subtracted from
the values of the sham limb to yield a net
difference and were recorded every 5 min
continuously on time course graphs [25]
25 Intravital Microscopy
Male SD rats were anesthetized with 60
mgkg pentobarbital sodium IP and maintained
with 30 mgkg pentobarbital IP The left carotid
artery was isolated and a PE-50 polyethylene
catheter was inserted in the left carotid artery for
monitoring of the mean arterial blood pressure
(MABP) via BP-1 Pressure Monitor (WPI Inc
Sarasota FL) After abdominal laparotomy a
loop of ileal mesentery was exteriorized and
placed in a temperature controlled Plexiglas
chamber (37 degC) for adequate superfusion of the
test solutions [27] The mesentery was placed
over a Plexiglas pedestal in the observation
chamber for visualization under a Nikon Eclipse
microscope (Nikon Co Japan) The
microcirculation was recorded with Image Pro
MDA (Media Cybernetics Bethesda MD) and
leukocyte-endothelial interactions were analyzed
offline
The rats were allowed to stabilize for 30 min
with superfusion of Krebsrsquo buffer after surgery
After stabilization a non-branched postcapillary
venule was chosen for observation An initial
recording was made to establish basal values for
leukocyte rolling adherence and transmigration
The mesentery then was superfused with the
experimental test solutions for 120 min Test
solutions included Krebsrsquo buffer alone 50 M L-
NAME alone and 50 M L-NAME in the
presence of Nox2ds-tat (5 M or 20 M) All
drugs were dissolved into the Krebsrsquo buffer The
solution was aerated with 95 N2-5 CO2 and
equilibrated at a pH of 73 to 74 at 37 degC Two
min video recordings were made at baseline 30
min 60 min 90 min and 120 min after
superfusion of the test solutions for quantification
of leukocyte rolling adherence and
transmigration [24 27]
26 Hematoxylin and Eosin (HampE) Staining After the experiment the loop of the ileal
mesentery that was superfused during the
experiment was removed and quickly stored in 4
paraformaldehyde for histological analysis of
leukocyte adherence and transmigration by HampE
staining Three representative sections of the ileal
mesentery from Krebsrsquo buffer 50 microM L-NAME
and 50 microM L-NAME in the presence of Nox2ds-
tat (5 M or 20 microM) were selected for
histological analysis Tissue samples were
selected from experiments representative of the
group mean of intravital microscopy (Table 2)
The tissue was embedded in paraffin and
sectioned into 45 microm serial sections and placed
onto glass slides Sections were deparaffinized
and rehydrated then stained with HampE [27]
Under light microscopy leukocyte adherence and
transmigration was counted in areas containing
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 214
venulesarterioles within the serosa and the
mesentery and expressed as adhered and
transmigrated leukocytesmm2 in tissue
27 Statistical Analysis
All data in the text and figures are presented
as means plusmn standard error of the mean (SEM)
The data was analyzed by analysis of variance
(ANOVA) using post hoc analysis with the
Student-Newman-Keuls test for all experiments
except for hindlimb IR in which a studentrsquos t-test
was used A p value of lt005 was considered to
be statistically significant
3 Results
31 PMN SO release
PMA is a broad-spectrum activator of protein
kinase C (PKC) which is essential in promoting
NADPH oxidase assembly at the PMN
membrane to generate SO release [28]We found
that Nox2ds-tat exhibited a dose-dependent
reduction in PMA-stimulated PMN SO release
(Figure 3) Doses of Nox2ds-tat (10 μM and 40
μM) did not significantly decrease PMN SO
release whereas a dose of 80 μM significantly
inhibited PMA-induced SO release by 37 plusmn 7
(plt005 compared to PMA alone) The data
generated from this assay was used to test a
similar dose range of Nox2ds-tat in isolated rat
heart IR experiments
Figure 3 PMA (100 nM) induced SO release in PMNs peak response read at 360s post stimulation Nox2ds-tat dose
dependently inhibited SO release from PMNs by 37 plusmn 7 (80 microM n=4) when compared to PMNs treated with PMA
alone (plt005 vs PMA control)
32 Cardiac function and infarct size
Nox2ds-tat given at reperfusion dose-
dependently attenuated IR induced cardiac
contractile dysfunction and recovered post-
reperfused LVDP to 47 plusmn 7 (5 μM n=7) 69 plusmn
13 (10 μM n=6 plt005) 68 plusmn 7 (40 μM
n=6 plt005) and 77 plusmn 7 (80 μM n=6 plt005)
of initial LVDP at 45 min post-reperfusion when
compared to control IR hearts that only
recovered to 46 plusmn 6 (n=14) (Table 1) Initial
baseline values for cardiac function parameters
did not differ significantly among groups
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 215
Table 1 Cardiac function parameters among different experimental groups
Cardiac Parameters Control IR
(n=14)
IR +
Nox2ds-tat 5
μM
(n=7)
IR +
Nox2ds-tat
10 μM
(n=6)
IR +
Nox2ds-tat
40 μM
(n=6)
IR +
Nox2ds-tat
80 μM
(n=6)
Initial LVDP (mmHg) 87plusmn35 91plusmn31 89plusmn34 92plusmn39 88plusmn33
Final LVDP (mmHg) 40plusmn50 43plusmn66 61plusmn121 63plusmn62 68plusmn62
Initial LVESP (mmHg) 99plusmn38 101plusmn39 105plusmn48 103plusmn46 101plusmn37
Final LVESP (mmHg) 100plusmn53 108plusmn57 107plusmn119 110plusmn75 124plusmn97
Initial LVEDP (mmHg) 11plusmn10 94plusmn13 16plusmn20 11plusmn13 13plusmn07
Final LVEDP(mmHg) 61plusmn51 64plusmn42 46plusmn61 47plusmn75
56plusmn56
Initial +dPdtmax (mmHgsec) 2259plusmn71 2335plusmn61 2333plusmn66 2346plusmn77 2277plusmn79
Final +dPdtmax (mmHgsec) 894plusmn105 832plusmn124 1285plusmn232 1258plusmn120
1413plusmn100
Initial -dPdtmin (mmHgsec) -1510plusmn67 -1616plusmn98 -1607plusmn84 -1550plusmn124 -1524plusmn106
Final -dPdtmin (mmHgsec) -704plusmn65 -660plusmn136 -879plusmn187 -939plusmn108 -1116plusmn146
Initial coronary flow
(mlmin) 162plusmn12 144plusmn064 181plusmn14 180plusmn25 152plusmn10
Final coronary flow (mlmin) 79plusmn064 79plusmn070 92plusmn15 93plusmn17 95plusmn15
Initial Heart Rate (BPM) 271plusmn128 277plusmn123 280plusmn78 272plusmn88 265plusmn32
Final Heart Rate (BPM) 246plusmn188 234plusmn199 230plusmn105 249plusmn136 237plusmn119
plt005 vs control IR final cardiac function parameters plt005
plt001 vs Nox2ds-tat 5μM
Similarly Nox2ds-tat reduced infarct
sizearea-at-risk when compared to the IR
control group (Figure 4 Panel A-B) The cross
section for the IR control group shows the
majority of the infarct in the mid-wall area
whereas the Nox2ds-tat treated IR hearts showed
less infarct area (Figure 4 Panel A) Figure 4
Panel B shows the ratio of this tissue at risk
When compared to the IR control group (46 plusmn
2) there was a significant decrease in total
infarct size in hearts treated with Nox2ds-tat 30 plusmn
4 (5 μM) 15 plusmn 14 (10 μM) 23 plusmn 2 (40
microM) and 19 plusmn 16 (80 microM)(plt001 for all
groups) Moreover the 10 μM and 80 μM doses
showed significantly less infarct size compared to
the 5 μM dose (plt001 and plt005 respectively)
suggesting a dose-dependent effect of Nox2ds-tat
on infarct size (Figure 4 Panel A-B)
Other cardiac function parameters are
illustrated in Table 1 Of interest Nox2ds-tat
dose-dependently recovered post-reperfused final
+dPdtmax by 36 plusmn 53 (5 μM) 55 plusmn10 (10
μM) 54 plusmn 4 (40 microM plt005) 62 plusmn 4 (80 microM
plt005) at 45 min reperfusion compared to
control IR hearts that only recovered to 40 plusmn 5
Sham hearts (n=6) were not subjected to IR and
maintained cardiac function parameters and flow
by gt90 and had lt5 infarct sizearea-at- risk
throughout the experimental time-course (data
not shown)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 216
Figure 4 Panel A Representative images of TTC staining and percentage of left ventricular heart tissue at risk among
different experimental groups Viable tissue stains red infarcted tissue did not retain TTC staining Nox2ds-tat dose
dependently attenuated infarct size Panel B Ratio of left ventricular heart tissue at risk for myocardial dysfunction
out of total tissue weight as determined by TTC staining of representative heart sections When compared to the IR
control group which had an infarct size of 46plusmn2 there was a significant decrease in total infarct size in heart sections
treated with Nox2ds-tat Nox2ds-tat dose-dependently attenuated infarct sizes to 30 plusmn 4 (5 μM n=7) 15 plusmn 14 (10
μM n=6) 23 plusmn 2 (40 microM n=6) and 19 plusmn 16 (80 microM n=6) (plt001 vs control tissue n=14) There was also a
significant decrease in infarct size observed in 10 μM (plt001) and 80 microM (plt005) compared to 5 μM Nox2ds-tat
(plt001 vs control IR plt005 vs Nox2ds-tat 5 microM plt001 vs Nox2ds-tat 5microM)
33 Hindlimb IR in vivo
331 Nox2ds-tat reduced blood H2O2 levels
The blood H2O2 values in the IR hindlimb of
saline controls significantly increased to 17 plusmn 03
microM (15 min post-reperfusion) above sham values
Thereafter a relative difference of 12-14 μM
between the IR and sham limbs was maintained
throughout the remainder of reperfusion (Figure 5
Panel A) By contrast Nox2ds-tat (41 mgkg
~20 μM in blood) treated animals in the IR
hindlimb did not significantly differ from the
sham hindlimb throughout reperfusion (Figure 5
Panel B) The relative difference in blood H2O2
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant reduction in the IR induced effect on
blood H2O2 by 143 plusmn 02 μM at the end of
reperfusion (Figure 5 Panel C)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 217
Figure 5 Panel A Relative difference in measured blood H2O2 between IR and sham femoral veins in saline controls
The saline IR vein showed a significant increase in blood H2O2 by 17 plusmn 03 microM (5 min) relative to the sham femoral
vein This increase above sham femoral vein values was maintained throughout reperfusion stabilizing at 14 plusmn 02 microM
(45 min) (plt005 plt001 vs sham IR femoral vein) Panel B Relative difference in measured blood H2O2
between IR and sham femoral veins in Nox2ds-tat (41 mgkg ~20 microM) treated rats The IR femoral vein treated with
Nox2ds-tat showed a transient increase in H2O2 when compared to the sham femoral vein However this difference
between the two limbs decreased throughout the remainder of the experiment ending at ~0 microM The measured values
in the two groups were not statistically different at any point in the experiment Panel C Relative difference in
measured blood H2O2 betweenNox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline
values) Nox2ds-tat infusion at reperfusion significantly decreased the blood H2O2 concentration when compared to
rats infused with saline At the end of the 45 min reperfusion period there was a significant reduction of 14 microM
between blood H2O2 measured in Nox2ds-tat 20 microM treated rats relative to saline control (plt005 plt001 vs
saline treated femoral veins)
B
A
C
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 218
A
B
C
Figure 6 Panel A Relative difference in measured blood NO between IR and sham femoral veins in saline controls
Overall the IR femoral vein showed a significant decrease in NO concentration stabilizing at a difference of 90 plusmn 21
nM when compared to the sham femoral vein at the end of the 45 min reperfusion period (plt005 plt001 vs sham
IR femoral vein) Panel B Relative difference in measured blood NO between IR and sham femoral veins in Nox2ds-
tat (41 mgkg ~20 microM) treated rats In Nox2ds-tat treated animals the IR femoral vein resulted in 37 plusmn 12 nM higher
blood NO concentration than the sham limb at the end of the 45 min reperfusion period however this difference was
not statistically significant (plt005 plt001 vs sham IR femoral vein) Panel C Relative difference in measured
blood NO in Nox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline values) Nox2ds-tat
given at reperfusion significantly increased the total blood NO concentration when compared to saline controls At the
end of the 45 min reperfusion period there was a significant increase of 127 nM in blood NO in Nox2ds-tat treated
animals relative to saline controls (plt005 plt001 vs saline treated femoral veins)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 219
332 Nox2ds-tat prevented the decrease in
blood NO levels
In saline controls blood NO levels steadily
decreased in the IR limb throughout the
reperfusion period leading to significantly lower
blood NO bioavailability (90 nM below sham) as
early as 20 min into the reperfusion period
(Figure 6 Panel A) By contrast blood NO in the
IR hindlimb of Nox2ds-tat (41 mgkg ~20 μM
in blood) treated animals did not significantly
differ from sham hindlimb values during the
reperfusion period While a linear trend close to
sham values was observed the Nox2ds-tat treated
grouprsquos blood NO levels did rise to 37 nM above
sham values at the end of reperfusion (Figure 6
Panel B) The relative difference in blood NO
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant increase in the IR induced effect on
NO release by 127 nM at the end of reperfusion
(Figure 6 Panel C)
34 L-NAME induced Leukocyte endothelial
interactions
341 Intravital Microscopy
There was no significant difference among
initial baseline values in leukocyte rolling
adherence and transmigration among all study
groups In all parameters the Krebsrsquo buffer group
did not significantly change from baseline L-
NAME (50 microM) significantly increased leukocyte
rolling adherence and transmigration when
compared to Krebsrsquo at T=120 min by 82 plusmn 11
(plt001) 22 plusmn 5 (plt001) and 21 plusmn 4 (plt001)
respectively (Table 2) Nox2ds-tat (5 microM) did not
significantly attenuate L-NAME induced
leukocyte-endothelial interactions except for final
transmigration (plt005) By contrast Nox2ds-tat
(20 microM) significantly decreased the L-NAME
induced effect by 3-5 fold on all parameters
(plt001) (Table 2)
Table 2 The comparision of initial and final (120 min of reperfusion) leukocyte rolling adherence and transmigration
between Krebsrsquo L-NAME and L-NAME + Nox2ds-tat (5 μM 20 μM) groups in rat mesenteric venules
Intravital microscopy Parameters
Krebsrsquo
(n=6)
L-NAME
(n=5)
L-NAME +
Nox2ds-tat 5 μM
(n=4)
L-NAME +
Nox2ds-tat 20 μM
(n=6)
Initial Rolling (numbermin) 8plusmn3 11plusmn3 13plusmn3 8plusmn2
Final Rolling (numbermin) 16plusmn5 82plusmn11
72plusmn13 16plusmn6++
Initial Adherence (number100um) 2plusmn2 1plusmn0 3plusmn0 1plusmn1
Final Adherence (number100um) 4plusmn2 22plusmn5 17plusmn1 8plusmn4
Initial Transmigration (number) 1plusmn0 1plusmn0 1plusmn0 1plusmn0
Final Transmigration (number) 1plusmn0 21plusmn4 9plusmn1
4plusmn2
Plt005 Plt001 compared to Krebsrsquo groupPlt005
Plt001 compared to L-NAME
++Plt001 compared to L-
NAME + Nox2ds-tat 5 μM
342 HampE Staining
The Krebsrsquocontrol group exhibited only 73 plusmn
33 and 62 plusmn 30 leukocytesmm2 of mesenteric
tissue These values represent basal numbers of
leukocyte adherence and transmigration in this
tissue bed respectively L-NAME treatment
exhibited a marked increase (~4-fold) in
leukocyte vascular adherence and transmigration
compared to Krebsrsquo buffer (299 plusmn 23 and 271 plusmn
26 leukocytesmm2 of tissue respectively plt001
for both values) Nox2ds-tat dose-dependently
attenuated the L-NAME induced effect on
leukocyte adherence (199 plusmn 13 for 5 microM at
plt005 and 83 plusmn 9 for 20 microM plt001) and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
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G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
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101161CIRCULATIONAHA113002714
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M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
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R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
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infarction Biomaterials 34(31) 7790-7798
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17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
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1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
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Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
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101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
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triumphs and pitfalls Antioxidants amp Redox
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101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
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10116101RES0000063423946458A
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Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
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phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
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B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
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249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
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1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
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27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
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Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
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101111j1527-34662005tb00170x
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Mechanisms for the role of
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R M (1994) Role of the membrane cortex
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inhibition attenuates myocardial infarction
induced heart failure and is associated with a
reduction of fibrosis and pro‐inflammatory
responses Journal of Cellular and
Molecular Medicine 15(8) 1769-1777 DOI
101111j1582-4934201001174x
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 210
minutes of reperfusion augmenting inflammation
associated injury [5-9] It is evident that NADPH
oxidase is a significant source of oxidative stress
during reperfusion both directly by producing
ROS and indirectly by propagating mediators of
IR injury
Figure 1 IR insult initially leads to cytokine mediated receptor stimulation followed by the activation of Nox2
which in turn produces SO The SO released from Nox2 induces additional ROS release from xanthine oxidase eNOS
uncoupling and dysfunctional mitochondria Collectively these sources of oxidative stress overproduce SOH2O2
species and diminish the bioavailability of blood NO This cyclical process of oxidative injury can promote further
mitochondrial dysfunction additional eNOS uncoupling and stimulate xanthine oxidase The aggregate response to
this oxidative stress at reperfusion is endothelial dysfunction leukocyte recruitment cardiomyocyte death and
increased ROS production all which lead to additional myocardial IR injury
The NADPH oxidase family contains seven
members among which Nox2 has been suggested
as an important isoform during myocardial IR
[10] First Nox2 is a ubiquitous enzyme within
several areas of the cardiovascular system such
as endothelial cells adventitial fibroblasts
myocytes and platelets and is the primary source
of SO production in Polymorphonuclear
Leukocytes (PMNs) Secondly Nox2 is located
on the cell membranes and subcellular organelles
of the aforementioned cells such as the
endoplasmic reticulum or nuclear membrane The
distribution of Nox2 allows it to affect
mitochondria and eNOS coupling in multiple
areas of the cell [11] Third Nox2 subunits can
be up-regulated by IR related mediators such as
inflammatory cytokines [12 13] Fourth Nox2
can sense the disturbance of blood flow during
periods of ischemia (ie oscillatory and eddy
current) and in response initiates vascular
inflammation which can exacerbate the initial
ischemic event [14] Lastly Nox2 deletion may
improve the cardiomyocyte survival pathway
such as the SAFE signaling pathway (ie
phosphorylation of STAT3) [15] The increase in
Nox2 activity within the cardiovascular
endothelium and leukocytes produces a
simultaneous release of SO and quenching of NO
(ie formation of peroxynitrite anion) resulting
in vascular oxidative stress endothelial
dysfunction and inflammation Studies have
shown that cardiac Nox2 knockout mice or the
delivery of Nox2 siRNA to the heart to suppress
heart Nox2 expression results in a significant
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 211
reduction of ROS production and final infarct
size in addition to improved cardiac function
following IR [16] These results are highly
suggestive that Nox2 is the primary source of SO
release in cardiac tissue
Despite previous research there are still no
published reports of any selective
pharmacological tools that address the effects of
Nox2 in IR injury and inflammation Research
involving apocynin a putative Nox2 inhibitor
was shown to exert cardioprotective effects
during reperfusion however this alkaloid of
Apocynum cannabinum can also exert direct anti-
oxidant effects independent of Nox2 inhibition
[10 17 18] Consequently our research group
chose to examine Nox2ds-tat (formerly known as
gp91ds-tat) a more selective Nox2 peptide
inhibitor to better understand the specific role of
Nox2 in propagating oxidative stress during IR
injury The tat portion of this peptide contains a
9-aa sequence (ie [H]- R-K-K-R-R-Q-R-R-R )
which facilitates Nox2ds-tat delivery into the cell
The docking sequence (ie C-S-T-R-R-R-Q-Q-
L-NH2) specifically inhibits the assembly of
cytosolic subunit p47phox
with the membrane
subunit gp91 and subsequently inhibits Nox2
assembly and activation (Figure 2) [19 20 21]
Moreover in recent studies Nox2ds-tat has been
shown to suppress balloon angioplasty induced
SO production in carotid artery ischemia
suggesting that the effects of Nox2ds-tat could be
extended to cardiomyocyte IR injury [21 22] It
is evident that Nox2ds-tat is a promising NADPH
oxidase inhibitor selectively targeting Nox2
which prevents the deleterious cycle of SO
production and NO depletion characteristic of
reperfusion-induced myocardial injury
Figure 2 Schematic of Nox2ds-tat mechanism of action whereby the blue line shows inhibition of cytosolic subunit
p47phox on the gp91 Nox2 membrane component This disallows for NADPH oxidase to assemble and the inherent
production of ROS with this event as depicted with the blue cross out Adapted from Wilkinson et al [40]
In this study we first determined the dose-
dependent effects of Nox2ds-tat on leukocyte SO
production Next the effects of this peptide were
evaluated in myocardial and hindlimb IR by
post-reperfused cardiac functioninfarct size and
NOH2O2 respectively We also studied the
effects of the peptide on NG-L-arginine methyl
ester (L-NAME) induced leukocyte-endothelial
interactions in rat mesenteric venules by intravital
microscopy We predict that Nox2ds-tat will
attenuate ROS in phorbol 12-myristate 13-acetate
(PMA) induced leukocytes and hindlimb IR will
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 212
decrease infarct size in myocardial IR and will
attenuate leukocyte-endothelial interactions in rat
mesenteric venules Moreover we predict that the
reduction in infarct size will parallel an
improvement in post reperfusion cardiac function
2 Methods
21 Animals
All animal procedures were approved by the
Institutional Animal Care and Use Committee at
PCOM for care and use of animals Male Sprague
Dawley (SD) rats (275-325 g Charles River
Springfield MA) were used for ex vivo and in
vivo experiments and 350-400 g male SD rats
were used for SO release assays listed below
22 Measurement of SO Release from Rat
PMNs
PMNs were isolated from male SD rats and
SO release was measured spectrophotometrically
(model 260 Gilford Nova Biotech El Cajon CA)
by the reduction of ferricytochrome c [23] The
PMNs (5x106) were re-suspended in 450 μl PBS
and incubated with ferricytochrome c (100 microM
Sigma Chemical) in a total volume of 900 μl PBS
in the presence or absence of varying
concentrations of Nox2ds-tat peptide inhibitor
([H]- R-K-K-R-R-Q-R-R-R-C-S-T-R-R-R-Q-Q-
L-NH2 MW=2452 gmol Genemed Synthesis
San Antoino TX) 10 M 40 M or 80 μM
respectively for 15 min at 37degC in
spectrophotometric cells The PMNs were
stimulated with 100 nM PMA (MW= 6168
gmol Calbiochem) in a final reaction volume of
10 ml Absorbance at 550 nm was measured
every 30 seconds for up to 360 seconds and the
change in absorbance (SO release) from PMNs
was determined relative to time 0 The dose-
response curve of Nox2ds-tat on PMN SO release
was used to determine a dose range that may be
effective in myocardial IR experiments
23 Isolated Rat Heart Preparation
Male SD rats were anesthetized
intraperitoneally (IP) with 60 mgkg pentobarbital
sodium and anticoagulated with 1000 U of
sodium heparin IP Plasma was prepared from
blood isolated from the abdominal aorta of the
same rat from which the heart was isolated from
for each cardiac perfusion experiment The
plasma was used as the vehicle for infusion of
Nox2ds-tat The hearts were rapidly excised and
perfused via the Langendorff heart preparation
[24] The aorta was cannulated and retrograde
perfused with modified Krebsrsquo buffer containing
170 mM dextrose 1200 mM NaCl 250 mM
NaHCO3 25 mM CaCl2 05 mM EDTA 59
mM KCl and 12 mM MgCl2 The modified
Krebsrsquo buffer was maintained at 37oC 80 mmHg
constant pressure aerated with 95 O2-5 CO2
and equilibrated pH between 73-74 Hearts were
immersed in an H2O jacket reservoir containing
160 ml of modified Krebsrsquo buffer at 37oC to
provide the preload The preload was established
by the volume of modified Krebsrsquo buffer that
entered the left ventricle The pressure transducer
catheter was inserted into the left ventricle of the
heart through the mitral valve Hearts were
subjected to 15 min Krebsrsquo buffer perfusion for
stabilization in order to record an initial baseline
then 30 min of global ischemia by stopping
perfusion followed by a 45 min reperfusion
period A volume of 5 ml of plasma (control) or
plasma containing Nox2ds-tat (5 μM 10 μM 40
μM or 80 μM) was injected during the first 5 min
of reperfusion by a side arm line proximal to the
heart inflow cannula at a rate of 1 mlmin All
cardiac function including coronary flow left
ventricular developed pressure ([LVDP] which is
the left ventricular end-systolic pressure [LVESP]
minus left ventricular end-diastolic pressure
[LVEDP]) maximal and minimal rate of LVDP
(+dPdtmax and ndashdPdtmin) and heart rate readings
(BPM) were taken every 5 min from initial
baseline and then throughout reperfusion
Coronary flow (mlmin) was recorded using an
inline flow meter (T106 Transonic Systems Inc
Ithaca NY) and LVDP LVESP LVEDP
+dPdtmax ndashdPdtmin and BPM readings were
recorded using a pressure transducer (SPR-524
Millar Instruments Inc Houston TX) Data was
recorded using a Powerlab Station acquisition
system (ADInstruments Grand Junction CO) At
the end of the experiment the left ventricle was
isolated and cross sectioned into 2mm pieces
from apex to base and was subjected to 1
triphenyltetrazolium chloride (TTC) staining for
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 213
15 min at 37oC to detect infarct size as previously
described [24]
24 Hindlimb IR in vivo procedure
We measured blood H2O2NO release in real-
time from sensors placed in the femoral veins of
male SD rats anesthetized with sodium
pentobarbital (60 mgkg IP for induction and 30
mgkg IP for maintenance) Thereafter one limb
was subjected to IR while the other limb was
used as a non-ischemic sham control in the same
rat The calibrated H2O2 or NO microsensors
(100 microm World Precision Instruments [WPI] Inc
Sarasota FL) were connected to a free radical
analyzer (Apollo 4000 WPI Inc) and inserted
into a 24 gauge catheter placed inside each
femoral vein as previously described by Chen et
al [25] and reviewed by Young et al [26]
Ischemia in one hindlimb was induced by
clamping the femoral arteryvein for 30 min
followed by 45 min of reperfusion Nox2ds-tat
(41 mgkg prepared in saline ~20 μM in blood)
or saline (non-drug control group) was
administered at the beginning of reperfusion as a
bolus via the jugular vein We continuously
recorded and collected the H2O2 or NO release
data at 5 min intervals during a 15 min baseline
30 min ischemia and 45 min reperfusion period
The changes in H2O2 or NO release (in pA)
during IR are expressed as relative change to
initial readings Thereafter the values were
converted to the concentration of H2O2 (μM) or
NO (nM) after correction to the respective
calibration curves The values recorded from the
femoral vein of the IR limb were subtracted from
the values of the sham limb to yield a net
difference and were recorded every 5 min
continuously on time course graphs [25]
25 Intravital Microscopy
Male SD rats were anesthetized with 60
mgkg pentobarbital sodium IP and maintained
with 30 mgkg pentobarbital IP The left carotid
artery was isolated and a PE-50 polyethylene
catheter was inserted in the left carotid artery for
monitoring of the mean arterial blood pressure
(MABP) via BP-1 Pressure Monitor (WPI Inc
Sarasota FL) After abdominal laparotomy a
loop of ileal mesentery was exteriorized and
placed in a temperature controlled Plexiglas
chamber (37 degC) for adequate superfusion of the
test solutions [27] The mesentery was placed
over a Plexiglas pedestal in the observation
chamber for visualization under a Nikon Eclipse
microscope (Nikon Co Japan) The
microcirculation was recorded with Image Pro
MDA (Media Cybernetics Bethesda MD) and
leukocyte-endothelial interactions were analyzed
offline
The rats were allowed to stabilize for 30 min
with superfusion of Krebsrsquo buffer after surgery
After stabilization a non-branched postcapillary
venule was chosen for observation An initial
recording was made to establish basal values for
leukocyte rolling adherence and transmigration
The mesentery then was superfused with the
experimental test solutions for 120 min Test
solutions included Krebsrsquo buffer alone 50 M L-
NAME alone and 50 M L-NAME in the
presence of Nox2ds-tat (5 M or 20 M) All
drugs were dissolved into the Krebsrsquo buffer The
solution was aerated with 95 N2-5 CO2 and
equilibrated at a pH of 73 to 74 at 37 degC Two
min video recordings were made at baseline 30
min 60 min 90 min and 120 min after
superfusion of the test solutions for quantification
of leukocyte rolling adherence and
transmigration [24 27]
26 Hematoxylin and Eosin (HampE) Staining After the experiment the loop of the ileal
mesentery that was superfused during the
experiment was removed and quickly stored in 4
paraformaldehyde for histological analysis of
leukocyte adherence and transmigration by HampE
staining Three representative sections of the ileal
mesentery from Krebsrsquo buffer 50 microM L-NAME
and 50 microM L-NAME in the presence of Nox2ds-
tat (5 M or 20 microM) were selected for
histological analysis Tissue samples were
selected from experiments representative of the
group mean of intravital microscopy (Table 2)
The tissue was embedded in paraffin and
sectioned into 45 microm serial sections and placed
onto glass slides Sections were deparaffinized
and rehydrated then stained with HampE [27]
Under light microscopy leukocyte adherence and
transmigration was counted in areas containing
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 214
venulesarterioles within the serosa and the
mesentery and expressed as adhered and
transmigrated leukocytesmm2 in tissue
27 Statistical Analysis
All data in the text and figures are presented
as means plusmn standard error of the mean (SEM)
The data was analyzed by analysis of variance
(ANOVA) using post hoc analysis with the
Student-Newman-Keuls test for all experiments
except for hindlimb IR in which a studentrsquos t-test
was used A p value of lt005 was considered to
be statistically significant
3 Results
31 PMN SO release
PMA is a broad-spectrum activator of protein
kinase C (PKC) which is essential in promoting
NADPH oxidase assembly at the PMN
membrane to generate SO release [28]We found
that Nox2ds-tat exhibited a dose-dependent
reduction in PMA-stimulated PMN SO release
(Figure 3) Doses of Nox2ds-tat (10 μM and 40
μM) did not significantly decrease PMN SO
release whereas a dose of 80 μM significantly
inhibited PMA-induced SO release by 37 plusmn 7
(plt005 compared to PMA alone) The data
generated from this assay was used to test a
similar dose range of Nox2ds-tat in isolated rat
heart IR experiments
Figure 3 PMA (100 nM) induced SO release in PMNs peak response read at 360s post stimulation Nox2ds-tat dose
dependently inhibited SO release from PMNs by 37 plusmn 7 (80 microM n=4) when compared to PMNs treated with PMA
alone (plt005 vs PMA control)
32 Cardiac function and infarct size
Nox2ds-tat given at reperfusion dose-
dependently attenuated IR induced cardiac
contractile dysfunction and recovered post-
reperfused LVDP to 47 plusmn 7 (5 μM n=7) 69 plusmn
13 (10 μM n=6 plt005) 68 plusmn 7 (40 μM
n=6 plt005) and 77 plusmn 7 (80 μM n=6 plt005)
of initial LVDP at 45 min post-reperfusion when
compared to control IR hearts that only
recovered to 46 plusmn 6 (n=14) (Table 1) Initial
baseline values for cardiac function parameters
did not differ significantly among groups
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 215
Table 1 Cardiac function parameters among different experimental groups
Cardiac Parameters Control IR
(n=14)
IR +
Nox2ds-tat 5
μM
(n=7)
IR +
Nox2ds-tat
10 μM
(n=6)
IR +
Nox2ds-tat
40 μM
(n=6)
IR +
Nox2ds-tat
80 μM
(n=6)
Initial LVDP (mmHg) 87plusmn35 91plusmn31 89plusmn34 92plusmn39 88plusmn33
Final LVDP (mmHg) 40plusmn50 43plusmn66 61plusmn121 63plusmn62 68plusmn62
Initial LVESP (mmHg) 99plusmn38 101plusmn39 105plusmn48 103plusmn46 101plusmn37
Final LVESP (mmHg) 100plusmn53 108plusmn57 107plusmn119 110plusmn75 124plusmn97
Initial LVEDP (mmHg) 11plusmn10 94plusmn13 16plusmn20 11plusmn13 13plusmn07
Final LVEDP(mmHg) 61plusmn51 64plusmn42 46plusmn61 47plusmn75
56plusmn56
Initial +dPdtmax (mmHgsec) 2259plusmn71 2335plusmn61 2333plusmn66 2346plusmn77 2277plusmn79
Final +dPdtmax (mmHgsec) 894plusmn105 832plusmn124 1285plusmn232 1258plusmn120
1413plusmn100
Initial -dPdtmin (mmHgsec) -1510plusmn67 -1616plusmn98 -1607plusmn84 -1550plusmn124 -1524plusmn106
Final -dPdtmin (mmHgsec) -704plusmn65 -660plusmn136 -879plusmn187 -939plusmn108 -1116plusmn146
Initial coronary flow
(mlmin) 162plusmn12 144plusmn064 181plusmn14 180plusmn25 152plusmn10
Final coronary flow (mlmin) 79plusmn064 79plusmn070 92plusmn15 93plusmn17 95plusmn15
Initial Heart Rate (BPM) 271plusmn128 277plusmn123 280plusmn78 272plusmn88 265plusmn32
Final Heart Rate (BPM) 246plusmn188 234plusmn199 230plusmn105 249plusmn136 237plusmn119
plt005 vs control IR final cardiac function parameters plt005
plt001 vs Nox2ds-tat 5μM
Similarly Nox2ds-tat reduced infarct
sizearea-at-risk when compared to the IR
control group (Figure 4 Panel A-B) The cross
section for the IR control group shows the
majority of the infarct in the mid-wall area
whereas the Nox2ds-tat treated IR hearts showed
less infarct area (Figure 4 Panel A) Figure 4
Panel B shows the ratio of this tissue at risk
When compared to the IR control group (46 plusmn
2) there was a significant decrease in total
infarct size in hearts treated with Nox2ds-tat 30 plusmn
4 (5 μM) 15 plusmn 14 (10 μM) 23 plusmn 2 (40
microM) and 19 plusmn 16 (80 microM)(plt001 for all
groups) Moreover the 10 μM and 80 μM doses
showed significantly less infarct size compared to
the 5 μM dose (plt001 and plt005 respectively)
suggesting a dose-dependent effect of Nox2ds-tat
on infarct size (Figure 4 Panel A-B)
Other cardiac function parameters are
illustrated in Table 1 Of interest Nox2ds-tat
dose-dependently recovered post-reperfused final
+dPdtmax by 36 plusmn 53 (5 μM) 55 plusmn10 (10
μM) 54 plusmn 4 (40 microM plt005) 62 plusmn 4 (80 microM
plt005) at 45 min reperfusion compared to
control IR hearts that only recovered to 40 plusmn 5
Sham hearts (n=6) were not subjected to IR and
maintained cardiac function parameters and flow
by gt90 and had lt5 infarct sizearea-at- risk
throughout the experimental time-course (data
not shown)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 216
Figure 4 Panel A Representative images of TTC staining and percentage of left ventricular heart tissue at risk among
different experimental groups Viable tissue stains red infarcted tissue did not retain TTC staining Nox2ds-tat dose
dependently attenuated infarct size Panel B Ratio of left ventricular heart tissue at risk for myocardial dysfunction
out of total tissue weight as determined by TTC staining of representative heart sections When compared to the IR
control group which had an infarct size of 46plusmn2 there was a significant decrease in total infarct size in heart sections
treated with Nox2ds-tat Nox2ds-tat dose-dependently attenuated infarct sizes to 30 plusmn 4 (5 μM n=7) 15 plusmn 14 (10
μM n=6) 23 plusmn 2 (40 microM n=6) and 19 plusmn 16 (80 microM n=6) (plt001 vs control tissue n=14) There was also a
significant decrease in infarct size observed in 10 μM (plt001) and 80 microM (plt005) compared to 5 μM Nox2ds-tat
(plt001 vs control IR plt005 vs Nox2ds-tat 5 microM plt001 vs Nox2ds-tat 5microM)
33 Hindlimb IR in vivo
331 Nox2ds-tat reduced blood H2O2 levels
The blood H2O2 values in the IR hindlimb of
saline controls significantly increased to 17 plusmn 03
microM (15 min post-reperfusion) above sham values
Thereafter a relative difference of 12-14 μM
between the IR and sham limbs was maintained
throughout the remainder of reperfusion (Figure 5
Panel A) By contrast Nox2ds-tat (41 mgkg
~20 μM in blood) treated animals in the IR
hindlimb did not significantly differ from the
sham hindlimb throughout reperfusion (Figure 5
Panel B) The relative difference in blood H2O2
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant reduction in the IR induced effect on
blood H2O2 by 143 plusmn 02 μM at the end of
reperfusion (Figure 5 Panel C)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 217
Figure 5 Panel A Relative difference in measured blood H2O2 between IR and sham femoral veins in saline controls
The saline IR vein showed a significant increase in blood H2O2 by 17 plusmn 03 microM (5 min) relative to the sham femoral
vein This increase above sham femoral vein values was maintained throughout reperfusion stabilizing at 14 plusmn 02 microM
(45 min) (plt005 plt001 vs sham IR femoral vein) Panel B Relative difference in measured blood H2O2
between IR and sham femoral veins in Nox2ds-tat (41 mgkg ~20 microM) treated rats The IR femoral vein treated with
Nox2ds-tat showed a transient increase in H2O2 when compared to the sham femoral vein However this difference
between the two limbs decreased throughout the remainder of the experiment ending at ~0 microM The measured values
in the two groups were not statistically different at any point in the experiment Panel C Relative difference in
measured blood H2O2 betweenNox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline
values) Nox2ds-tat infusion at reperfusion significantly decreased the blood H2O2 concentration when compared to
rats infused with saline At the end of the 45 min reperfusion period there was a significant reduction of 14 microM
between blood H2O2 measured in Nox2ds-tat 20 microM treated rats relative to saline control (plt005 plt001 vs
saline treated femoral veins)
B
A
C
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 218
A
B
C
Figure 6 Panel A Relative difference in measured blood NO between IR and sham femoral veins in saline controls
Overall the IR femoral vein showed a significant decrease in NO concentration stabilizing at a difference of 90 plusmn 21
nM when compared to the sham femoral vein at the end of the 45 min reperfusion period (plt005 plt001 vs sham
IR femoral vein) Panel B Relative difference in measured blood NO between IR and sham femoral veins in Nox2ds-
tat (41 mgkg ~20 microM) treated rats In Nox2ds-tat treated animals the IR femoral vein resulted in 37 plusmn 12 nM higher
blood NO concentration than the sham limb at the end of the 45 min reperfusion period however this difference was
not statistically significant (plt005 plt001 vs sham IR femoral vein) Panel C Relative difference in measured
blood NO in Nox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline values) Nox2ds-tat
given at reperfusion significantly increased the total blood NO concentration when compared to saline controls At the
end of the 45 min reperfusion period there was a significant increase of 127 nM in blood NO in Nox2ds-tat treated
animals relative to saline controls (plt005 plt001 vs saline treated femoral veins)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 219
332 Nox2ds-tat prevented the decrease in
blood NO levels
In saline controls blood NO levels steadily
decreased in the IR limb throughout the
reperfusion period leading to significantly lower
blood NO bioavailability (90 nM below sham) as
early as 20 min into the reperfusion period
(Figure 6 Panel A) By contrast blood NO in the
IR hindlimb of Nox2ds-tat (41 mgkg ~20 μM
in blood) treated animals did not significantly
differ from sham hindlimb values during the
reperfusion period While a linear trend close to
sham values was observed the Nox2ds-tat treated
grouprsquos blood NO levels did rise to 37 nM above
sham values at the end of reperfusion (Figure 6
Panel B) The relative difference in blood NO
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant increase in the IR induced effect on
NO release by 127 nM at the end of reperfusion
(Figure 6 Panel C)
34 L-NAME induced Leukocyte endothelial
interactions
341 Intravital Microscopy
There was no significant difference among
initial baseline values in leukocyte rolling
adherence and transmigration among all study
groups In all parameters the Krebsrsquo buffer group
did not significantly change from baseline L-
NAME (50 microM) significantly increased leukocyte
rolling adherence and transmigration when
compared to Krebsrsquo at T=120 min by 82 plusmn 11
(plt001) 22 plusmn 5 (plt001) and 21 plusmn 4 (plt001)
respectively (Table 2) Nox2ds-tat (5 microM) did not
significantly attenuate L-NAME induced
leukocyte-endothelial interactions except for final
transmigration (plt005) By contrast Nox2ds-tat
(20 microM) significantly decreased the L-NAME
induced effect by 3-5 fold on all parameters
(plt001) (Table 2)
Table 2 The comparision of initial and final (120 min of reperfusion) leukocyte rolling adherence and transmigration
between Krebsrsquo L-NAME and L-NAME + Nox2ds-tat (5 μM 20 μM) groups in rat mesenteric venules
Intravital microscopy Parameters
Krebsrsquo
(n=6)
L-NAME
(n=5)
L-NAME +
Nox2ds-tat 5 μM
(n=4)
L-NAME +
Nox2ds-tat 20 μM
(n=6)
Initial Rolling (numbermin) 8plusmn3 11plusmn3 13plusmn3 8plusmn2
Final Rolling (numbermin) 16plusmn5 82plusmn11
72plusmn13 16plusmn6++
Initial Adherence (number100um) 2plusmn2 1plusmn0 3plusmn0 1plusmn1
Final Adherence (number100um) 4plusmn2 22plusmn5 17plusmn1 8plusmn4
Initial Transmigration (number) 1plusmn0 1plusmn0 1plusmn0 1plusmn0
Final Transmigration (number) 1plusmn0 21plusmn4 9plusmn1
4plusmn2
Plt005 Plt001 compared to Krebsrsquo groupPlt005
Plt001 compared to L-NAME
++Plt001 compared to L-
NAME + Nox2ds-tat 5 μM
342 HampE Staining
The Krebsrsquocontrol group exhibited only 73 plusmn
33 and 62 plusmn 30 leukocytesmm2 of mesenteric
tissue These values represent basal numbers of
leukocyte adherence and transmigration in this
tissue bed respectively L-NAME treatment
exhibited a marked increase (~4-fold) in
leukocyte vascular adherence and transmigration
compared to Krebsrsquo buffer (299 plusmn 23 and 271 plusmn
26 leukocytesmm2 of tissue respectively plt001
for both values) Nox2ds-tat dose-dependently
attenuated the L-NAME induced effect on
leukocyte adherence (199 plusmn 13 for 5 microM at
plt005 and 83 plusmn 9 for 20 microM plt001) and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
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Myocardial reperfusion injury New England
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Myocardial ischemia-reperfusion injury a
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101172JCI62874
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Pathophysiological Roles of NADPH
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7 Fabian R H Perez-Polo J R amp Kent T
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Michael L H Youker K A Bressler R
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101089ars20135605
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101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
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(2011) Central TNF inhibition results in
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14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
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107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
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phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
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pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
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101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
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101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
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Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
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38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
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39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
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101795223APS2013064
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Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 211
reduction of ROS production and final infarct
size in addition to improved cardiac function
following IR [16] These results are highly
suggestive that Nox2 is the primary source of SO
release in cardiac tissue
Despite previous research there are still no
published reports of any selective
pharmacological tools that address the effects of
Nox2 in IR injury and inflammation Research
involving apocynin a putative Nox2 inhibitor
was shown to exert cardioprotective effects
during reperfusion however this alkaloid of
Apocynum cannabinum can also exert direct anti-
oxidant effects independent of Nox2 inhibition
[10 17 18] Consequently our research group
chose to examine Nox2ds-tat (formerly known as
gp91ds-tat) a more selective Nox2 peptide
inhibitor to better understand the specific role of
Nox2 in propagating oxidative stress during IR
injury The tat portion of this peptide contains a
9-aa sequence (ie [H]- R-K-K-R-R-Q-R-R-R )
which facilitates Nox2ds-tat delivery into the cell
The docking sequence (ie C-S-T-R-R-R-Q-Q-
L-NH2) specifically inhibits the assembly of
cytosolic subunit p47phox
with the membrane
subunit gp91 and subsequently inhibits Nox2
assembly and activation (Figure 2) [19 20 21]
Moreover in recent studies Nox2ds-tat has been
shown to suppress balloon angioplasty induced
SO production in carotid artery ischemia
suggesting that the effects of Nox2ds-tat could be
extended to cardiomyocyte IR injury [21 22] It
is evident that Nox2ds-tat is a promising NADPH
oxidase inhibitor selectively targeting Nox2
which prevents the deleterious cycle of SO
production and NO depletion characteristic of
reperfusion-induced myocardial injury
Figure 2 Schematic of Nox2ds-tat mechanism of action whereby the blue line shows inhibition of cytosolic subunit
p47phox on the gp91 Nox2 membrane component This disallows for NADPH oxidase to assemble and the inherent
production of ROS with this event as depicted with the blue cross out Adapted from Wilkinson et al [40]
In this study we first determined the dose-
dependent effects of Nox2ds-tat on leukocyte SO
production Next the effects of this peptide were
evaluated in myocardial and hindlimb IR by
post-reperfused cardiac functioninfarct size and
NOH2O2 respectively We also studied the
effects of the peptide on NG-L-arginine methyl
ester (L-NAME) induced leukocyte-endothelial
interactions in rat mesenteric venules by intravital
microscopy We predict that Nox2ds-tat will
attenuate ROS in phorbol 12-myristate 13-acetate
(PMA) induced leukocytes and hindlimb IR will
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 212
decrease infarct size in myocardial IR and will
attenuate leukocyte-endothelial interactions in rat
mesenteric venules Moreover we predict that the
reduction in infarct size will parallel an
improvement in post reperfusion cardiac function
2 Methods
21 Animals
All animal procedures were approved by the
Institutional Animal Care and Use Committee at
PCOM for care and use of animals Male Sprague
Dawley (SD) rats (275-325 g Charles River
Springfield MA) were used for ex vivo and in
vivo experiments and 350-400 g male SD rats
were used for SO release assays listed below
22 Measurement of SO Release from Rat
PMNs
PMNs were isolated from male SD rats and
SO release was measured spectrophotometrically
(model 260 Gilford Nova Biotech El Cajon CA)
by the reduction of ferricytochrome c [23] The
PMNs (5x106) were re-suspended in 450 μl PBS
and incubated with ferricytochrome c (100 microM
Sigma Chemical) in a total volume of 900 μl PBS
in the presence or absence of varying
concentrations of Nox2ds-tat peptide inhibitor
([H]- R-K-K-R-R-Q-R-R-R-C-S-T-R-R-R-Q-Q-
L-NH2 MW=2452 gmol Genemed Synthesis
San Antoino TX) 10 M 40 M or 80 μM
respectively for 15 min at 37degC in
spectrophotometric cells The PMNs were
stimulated with 100 nM PMA (MW= 6168
gmol Calbiochem) in a final reaction volume of
10 ml Absorbance at 550 nm was measured
every 30 seconds for up to 360 seconds and the
change in absorbance (SO release) from PMNs
was determined relative to time 0 The dose-
response curve of Nox2ds-tat on PMN SO release
was used to determine a dose range that may be
effective in myocardial IR experiments
23 Isolated Rat Heart Preparation
Male SD rats were anesthetized
intraperitoneally (IP) with 60 mgkg pentobarbital
sodium and anticoagulated with 1000 U of
sodium heparin IP Plasma was prepared from
blood isolated from the abdominal aorta of the
same rat from which the heart was isolated from
for each cardiac perfusion experiment The
plasma was used as the vehicle for infusion of
Nox2ds-tat The hearts were rapidly excised and
perfused via the Langendorff heart preparation
[24] The aorta was cannulated and retrograde
perfused with modified Krebsrsquo buffer containing
170 mM dextrose 1200 mM NaCl 250 mM
NaHCO3 25 mM CaCl2 05 mM EDTA 59
mM KCl and 12 mM MgCl2 The modified
Krebsrsquo buffer was maintained at 37oC 80 mmHg
constant pressure aerated with 95 O2-5 CO2
and equilibrated pH between 73-74 Hearts were
immersed in an H2O jacket reservoir containing
160 ml of modified Krebsrsquo buffer at 37oC to
provide the preload The preload was established
by the volume of modified Krebsrsquo buffer that
entered the left ventricle The pressure transducer
catheter was inserted into the left ventricle of the
heart through the mitral valve Hearts were
subjected to 15 min Krebsrsquo buffer perfusion for
stabilization in order to record an initial baseline
then 30 min of global ischemia by stopping
perfusion followed by a 45 min reperfusion
period A volume of 5 ml of plasma (control) or
plasma containing Nox2ds-tat (5 μM 10 μM 40
μM or 80 μM) was injected during the first 5 min
of reperfusion by a side arm line proximal to the
heart inflow cannula at a rate of 1 mlmin All
cardiac function including coronary flow left
ventricular developed pressure ([LVDP] which is
the left ventricular end-systolic pressure [LVESP]
minus left ventricular end-diastolic pressure
[LVEDP]) maximal and minimal rate of LVDP
(+dPdtmax and ndashdPdtmin) and heart rate readings
(BPM) were taken every 5 min from initial
baseline and then throughout reperfusion
Coronary flow (mlmin) was recorded using an
inline flow meter (T106 Transonic Systems Inc
Ithaca NY) and LVDP LVESP LVEDP
+dPdtmax ndashdPdtmin and BPM readings were
recorded using a pressure transducer (SPR-524
Millar Instruments Inc Houston TX) Data was
recorded using a Powerlab Station acquisition
system (ADInstruments Grand Junction CO) At
the end of the experiment the left ventricle was
isolated and cross sectioned into 2mm pieces
from apex to base and was subjected to 1
triphenyltetrazolium chloride (TTC) staining for
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 213
15 min at 37oC to detect infarct size as previously
described [24]
24 Hindlimb IR in vivo procedure
We measured blood H2O2NO release in real-
time from sensors placed in the femoral veins of
male SD rats anesthetized with sodium
pentobarbital (60 mgkg IP for induction and 30
mgkg IP for maintenance) Thereafter one limb
was subjected to IR while the other limb was
used as a non-ischemic sham control in the same
rat The calibrated H2O2 or NO microsensors
(100 microm World Precision Instruments [WPI] Inc
Sarasota FL) were connected to a free radical
analyzer (Apollo 4000 WPI Inc) and inserted
into a 24 gauge catheter placed inside each
femoral vein as previously described by Chen et
al [25] and reviewed by Young et al [26]
Ischemia in one hindlimb was induced by
clamping the femoral arteryvein for 30 min
followed by 45 min of reperfusion Nox2ds-tat
(41 mgkg prepared in saline ~20 μM in blood)
or saline (non-drug control group) was
administered at the beginning of reperfusion as a
bolus via the jugular vein We continuously
recorded and collected the H2O2 or NO release
data at 5 min intervals during a 15 min baseline
30 min ischemia and 45 min reperfusion period
The changes in H2O2 or NO release (in pA)
during IR are expressed as relative change to
initial readings Thereafter the values were
converted to the concentration of H2O2 (μM) or
NO (nM) after correction to the respective
calibration curves The values recorded from the
femoral vein of the IR limb were subtracted from
the values of the sham limb to yield a net
difference and were recorded every 5 min
continuously on time course graphs [25]
25 Intravital Microscopy
Male SD rats were anesthetized with 60
mgkg pentobarbital sodium IP and maintained
with 30 mgkg pentobarbital IP The left carotid
artery was isolated and a PE-50 polyethylene
catheter was inserted in the left carotid artery for
monitoring of the mean arterial blood pressure
(MABP) via BP-1 Pressure Monitor (WPI Inc
Sarasota FL) After abdominal laparotomy a
loop of ileal mesentery was exteriorized and
placed in a temperature controlled Plexiglas
chamber (37 degC) for adequate superfusion of the
test solutions [27] The mesentery was placed
over a Plexiglas pedestal in the observation
chamber for visualization under a Nikon Eclipse
microscope (Nikon Co Japan) The
microcirculation was recorded with Image Pro
MDA (Media Cybernetics Bethesda MD) and
leukocyte-endothelial interactions were analyzed
offline
The rats were allowed to stabilize for 30 min
with superfusion of Krebsrsquo buffer after surgery
After stabilization a non-branched postcapillary
venule was chosen for observation An initial
recording was made to establish basal values for
leukocyte rolling adherence and transmigration
The mesentery then was superfused with the
experimental test solutions for 120 min Test
solutions included Krebsrsquo buffer alone 50 M L-
NAME alone and 50 M L-NAME in the
presence of Nox2ds-tat (5 M or 20 M) All
drugs were dissolved into the Krebsrsquo buffer The
solution was aerated with 95 N2-5 CO2 and
equilibrated at a pH of 73 to 74 at 37 degC Two
min video recordings were made at baseline 30
min 60 min 90 min and 120 min after
superfusion of the test solutions for quantification
of leukocyte rolling adherence and
transmigration [24 27]
26 Hematoxylin and Eosin (HampE) Staining After the experiment the loop of the ileal
mesentery that was superfused during the
experiment was removed and quickly stored in 4
paraformaldehyde for histological analysis of
leukocyte adherence and transmigration by HampE
staining Three representative sections of the ileal
mesentery from Krebsrsquo buffer 50 microM L-NAME
and 50 microM L-NAME in the presence of Nox2ds-
tat (5 M or 20 microM) were selected for
histological analysis Tissue samples were
selected from experiments representative of the
group mean of intravital microscopy (Table 2)
The tissue was embedded in paraffin and
sectioned into 45 microm serial sections and placed
onto glass slides Sections were deparaffinized
and rehydrated then stained with HampE [27]
Under light microscopy leukocyte adherence and
transmigration was counted in areas containing
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 214
venulesarterioles within the serosa and the
mesentery and expressed as adhered and
transmigrated leukocytesmm2 in tissue
27 Statistical Analysis
All data in the text and figures are presented
as means plusmn standard error of the mean (SEM)
The data was analyzed by analysis of variance
(ANOVA) using post hoc analysis with the
Student-Newman-Keuls test for all experiments
except for hindlimb IR in which a studentrsquos t-test
was used A p value of lt005 was considered to
be statistically significant
3 Results
31 PMN SO release
PMA is a broad-spectrum activator of protein
kinase C (PKC) which is essential in promoting
NADPH oxidase assembly at the PMN
membrane to generate SO release [28]We found
that Nox2ds-tat exhibited a dose-dependent
reduction in PMA-stimulated PMN SO release
(Figure 3) Doses of Nox2ds-tat (10 μM and 40
μM) did not significantly decrease PMN SO
release whereas a dose of 80 μM significantly
inhibited PMA-induced SO release by 37 plusmn 7
(plt005 compared to PMA alone) The data
generated from this assay was used to test a
similar dose range of Nox2ds-tat in isolated rat
heart IR experiments
Figure 3 PMA (100 nM) induced SO release in PMNs peak response read at 360s post stimulation Nox2ds-tat dose
dependently inhibited SO release from PMNs by 37 plusmn 7 (80 microM n=4) when compared to PMNs treated with PMA
alone (plt005 vs PMA control)
32 Cardiac function and infarct size
Nox2ds-tat given at reperfusion dose-
dependently attenuated IR induced cardiac
contractile dysfunction and recovered post-
reperfused LVDP to 47 plusmn 7 (5 μM n=7) 69 plusmn
13 (10 μM n=6 plt005) 68 plusmn 7 (40 μM
n=6 plt005) and 77 plusmn 7 (80 μM n=6 plt005)
of initial LVDP at 45 min post-reperfusion when
compared to control IR hearts that only
recovered to 46 plusmn 6 (n=14) (Table 1) Initial
baseline values for cardiac function parameters
did not differ significantly among groups
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 215
Table 1 Cardiac function parameters among different experimental groups
Cardiac Parameters Control IR
(n=14)
IR +
Nox2ds-tat 5
μM
(n=7)
IR +
Nox2ds-tat
10 μM
(n=6)
IR +
Nox2ds-tat
40 μM
(n=6)
IR +
Nox2ds-tat
80 μM
(n=6)
Initial LVDP (mmHg) 87plusmn35 91plusmn31 89plusmn34 92plusmn39 88plusmn33
Final LVDP (mmHg) 40plusmn50 43plusmn66 61plusmn121 63plusmn62 68plusmn62
Initial LVESP (mmHg) 99plusmn38 101plusmn39 105plusmn48 103plusmn46 101plusmn37
Final LVESP (mmHg) 100plusmn53 108plusmn57 107plusmn119 110plusmn75 124plusmn97
Initial LVEDP (mmHg) 11plusmn10 94plusmn13 16plusmn20 11plusmn13 13plusmn07
Final LVEDP(mmHg) 61plusmn51 64plusmn42 46plusmn61 47plusmn75
56plusmn56
Initial +dPdtmax (mmHgsec) 2259plusmn71 2335plusmn61 2333plusmn66 2346plusmn77 2277plusmn79
Final +dPdtmax (mmHgsec) 894plusmn105 832plusmn124 1285plusmn232 1258plusmn120
1413plusmn100
Initial -dPdtmin (mmHgsec) -1510plusmn67 -1616plusmn98 -1607plusmn84 -1550plusmn124 -1524plusmn106
Final -dPdtmin (mmHgsec) -704plusmn65 -660plusmn136 -879plusmn187 -939plusmn108 -1116plusmn146
Initial coronary flow
(mlmin) 162plusmn12 144plusmn064 181plusmn14 180plusmn25 152plusmn10
Final coronary flow (mlmin) 79plusmn064 79plusmn070 92plusmn15 93plusmn17 95plusmn15
Initial Heart Rate (BPM) 271plusmn128 277plusmn123 280plusmn78 272plusmn88 265plusmn32
Final Heart Rate (BPM) 246plusmn188 234plusmn199 230plusmn105 249plusmn136 237plusmn119
plt005 vs control IR final cardiac function parameters plt005
plt001 vs Nox2ds-tat 5μM
Similarly Nox2ds-tat reduced infarct
sizearea-at-risk when compared to the IR
control group (Figure 4 Panel A-B) The cross
section for the IR control group shows the
majority of the infarct in the mid-wall area
whereas the Nox2ds-tat treated IR hearts showed
less infarct area (Figure 4 Panel A) Figure 4
Panel B shows the ratio of this tissue at risk
When compared to the IR control group (46 plusmn
2) there was a significant decrease in total
infarct size in hearts treated with Nox2ds-tat 30 plusmn
4 (5 μM) 15 plusmn 14 (10 μM) 23 plusmn 2 (40
microM) and 19 plusmn 16 (80 microM)(plt001 for all
groups) Moreover the 10 μM and 80 μM doses
showed significantly less infarct size compared to
the 5 μM dose (plt001 and plt005 respectively)
suggesting a dose-dependent effect of Nox2ds-tat
on infarct size (Figure 4 Panel A-B)
Other cardiac function parameters are
illustrated in Table 1 Of interest Nox2ds-tat
dose-dependently recovered post-reperfused final
+dPdtmax by 36 plusmn 53 (5 μM) 55 plusmn10 (10
μM) 54 plusmn 4 (40 microM plt005) 62 plusmn 4 (80 microM
plt005) at 45 min reperfusion compared to
control IR hearts that only recovered to 40 plusmn 5
Sham hearts (n=6) were not subjected to IR and
maintained cardiac function parameters and flow
by gt90 and had lt5 infarct sizearea-at- risk
throughout the experimental time-course (data
not shown)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 216
Figure 4 Panel A Representative images of TTC staining and percentage of left ventricular heart tissue at risk among
different experimental groups Viable tissue stains red infarcted tissue did not retain TTC staining Nox2ds-tat dose
dependently attenuated infarct size Panel B Ratio of left ventricular heart tissue at risk for myocardial dysfunction
out of total tissue weight as determined by TTC staining of representative heart sections When compared to the IR
control group which had an infarct size of 46plusmn2 there was a significant decrease in total infarct size in heart sections
treated with Nox2ds-tat Nox2ds-tat dose-dependently attenuated infarct sizes to 30 plusmn 4 (5 μM n=7) 15 plusmn 14 (10
μM n=6) 23 plusmn 2 (40 microM n=6) and 19 plusmn 16 (80 microM n=6) (plt001 vs control tissue n=14) There was also a
significant decrease in infarct size observed in 10 μM (plt001) and 80 microM (plt005) compared to 5 μM Nox2ds-tat
(plt001 vs control IR plt005 vs Nox2ds-tat 5 microM plt001 vs Nox2ds-tat 5microM)
33 Hindlimb IR in vivo
331 Nox2ds-tat reduced blood H2O2 levels
The blood H2O2 values in the IR hindlimb of
saline controls significantly increased to 17 plusmn 03
microM (15 min post-reperfusion) above sham values
Thereafter a relative difference of 12-14 μM
between the IR and sham limbs was maintained
throughout the remainder of reperfusion (Figure 5
Panel A) By contrast Nox2ds-tat (41 mgkg
~20 μM in blood) treated animals in the IR
hindlimb did not significantly differ from the
sham hindlimb throughout reperfusion (Figure 5
Panel B) The relative difference in blood H2O2
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant reduction in the IR induced effect on
blood H2O2 by 143 plusmn 02 μM at the end of
reperfusion (Figure 5 Panel C)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 217
Figure 5 Panel A Relative difference in measured blood H2O2 between IR and sham femoral veins in saline controls
The saline IR vein showed a significant increase in blood H2O2 by 17 plusmn 03 microM (5 min) relative to the sham femoral
vein This increase above sham femoral vein values was maintained throughout reperfusion stabilizing at 14 plusmn 02 microM
(45 min) (plt005 plt001 vs sham IR femoral vein) Panel B Relative difference in measured blood H2O2
between IR and sham femoral veins in Nox2ds-tat (41 mgkg ~20 microM) treated rats The IR femoral vein treated with
Nox2ds-tat showed a transient increase in H2O2 when compared to the sham femoral vein However this difference
between the two limbs decreased throughout the remainder of the experiment ending at ~0 microM The measured values
in the two groups were not statistically different at any point in the experiment Panel C Relative difference in
measured blood H2O2 betweenNox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline
values) Nox2ds-tat infusion at reperfusion significantly decreased the blood H2O2 concentration when compared to
rats infused with saline At the end of the 45 min reperfusion period there was a significant reduction of 14 microM
between blood H2O2 measured in Nox2ds-tat 20 microM treated rats relative to saline control (plt005 plt001 vs
saline treated femoral veins)
B
A
C
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 218
A
B
C
Figure 6 Panel A Relative difference in measured blood NO between IR and sham femoral veins in saline controls
Overall the IR femoral vein showed a significant decrease in NO concentration stabilizing at a difference of 90 plusmn 21
nM when compared to the sham femoral vein at the end of the 45 min reperfusion period (plt005 plt001 vs sham
IR femoral vein) Panel B Relative difference in measured blood NO between IR and sham femoral veins in Nox2ds-
tat (41 mgkg ~20 microM) treated rats In Nox2ds-tat treated animals the IR femoral vein resulted in 37 plusmn 12 nM higher
blood NO concentration than the sham limb at the end of the 45 min reperfusion period however this difference was
not statistically significant (plt005 plt001 vs sham IR femoral vein) Panel C Relative difference in measured
blood NO in Nox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline values) Nox2ds-tat
given at reperfusion significantly increased the total blood NO concentration when compared to saline controls At the
end of the 45 min reperfusion period there was a significant increase of 127 nM in blood NO in Nox2ds-tat treated
animals relative to saline controls (plt005 plt001 vs saline treated femoral veins)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 219
332 Nox2ds-tat prevented the decrease in
blood NO levels
In saline controls blood NO levels steadily
decreased in the IR limb throughout the
reperfusion period leading to significantly lower
blood NO bioavailability (90 nM below sham) as
early as 20 min into the reperfusion period
(Figure 6 Panel A) By contrast blood NO in the
IR hindlimb of Nox2ds-tat (41 mgkg ~20 μM
in blood) treated animals did not significantly
differ from sham hindlimb values during the
reperfusion period While a linear trend close to
sham values was observed the Nox2ds-tat treated
grouprsquos blood NO levels did rise to 37 nM above
sham values at the end of reperfusion (Figure 6
Panel B) The relative difference in blood NO
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant increase in the IR induced effect on
NO release by 127 nM at the end of reperfusion
(Figure 6 Panel C)
34 L-NAME induced Leukocyte endothelial
interactions
341 Intravital Microscopy
There was no significant difference among
initial baseline values in leukocyte rolling
adherence and transmigration among all study
groups In all parameters the Krebsrsquo buffer group
did not significantly change from baseline L-
NAME (50 microM) significantly increased leukocyte
rolling adherence and transmigration when
compared to Krebsrsquo at T=120 min by 82 plusmn 11
(plt001) 22 plusmn 5 (plt001) and 21 plusmn 4 (plt001)
respectively (Table 2) Nox2ds-tat (5 microM) did not
significantly attenuate L-NAME induced
leukocyte-endothelial interactions except for final
transmigration (plt005) By contrast Nox2ds-tat
(20 microM) significantly decreased the L-NAME
induced effect by 3-5 fold on all parameters
(plt001) (Table 2)
Table 2 The comparision of initial and final (120 min of reperfusion) leukocyte rolling adherence and transmigration
between Krebsrsquo L-NAME and L-NAME + Nox2ds-tat (5 μM 20 μM) groups in rat mesenteric venules
Intravital microscopy Parameters
Krebsrsquo
(n=6)
L-NAME
(n=5)
L-NAME +
Nox2ds-tat 5 μM
(n=4)
L-NAME +
Nox2ds-tat 20 μM
(n=6)
Initial Rolling (numbermin) 8plusmn3 11plusmn3 13plusmn3 8plusmn2
Final Rolling (numbermin) 16plusmn5 82plusmn11
72plusmn13 16plusmn6++
Initial Adherence (number100um) 2plusmn2 1plusmn0 3plusmn0 1plusmn1
Final Adherence (number100um) 4plusmn2 22plusmn5 17plusmn1 8plusmn4
Initial Transmigration (number) 1plusmn0 1plusmn0 1plusmn0 1plusmn0
Final Transmigration (number) 1plusmn0 21plusmn4 9plusmn1
4plusmn2
Plt005 Plt001 compared to Krebsrsquo groupPlt005
Plt001 compared to L-NAME
++Plt001 compared to L-
NAME + Nox2ds-tat 5 μM
342 HampE Staining
The Krebsrsquocontrol group exhibited only 73 plusmn
33 and 62 plusmn 30 leukocytesmm2 of mesenteric
tissue These values represent basal numbers of
leukocyte adherence and transmigration in this
tissue bed respectively L-NAME treatment
exhibited a marked increase (~4-fold) in
leukocyte vascular adherence and transmigration
compared to Krebsrsquo buffer (299 plusmn 23 and 271 plusmn
26 leukocytesmm2 of tissue respectively plt001
for both values) Nox2ds-tat dose-dependently
attenuated the L-NAME induced effect on
leukocyte adherence (199 plusmn 13 for 5 microM at
plt005 and 83 plusmn 9 for 20 microM plt001) and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
References
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Myocardial reperfusion injury New England
Journal of Medicine 357(11) 1121-1135
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2 Hausenloy D J amp Yellon D M (2013)
Myocardial ischemia-reperfusion injury a
neglected therapeutic target Journal of
Clinical Investigation 123(1) 92-100 DOI
101172JCI62874
3 Lefer A M amp Lefer D J (1996) The role
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6363(96)00073-9
4 Ago T Kuroda J Kamouchi M
Sadoshima J amp Kitazono T (2011)
Pathophysiological Roles of NADPH
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101253circjCJ-11-0388
5 Doughan A K Harrison D G amp Dikalov
S I (2008) Molecular Mechanisms of
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488-496 DOI
101161CIRCRESAHA107162800
6 Duda M Konior A Klemenska E amp
Beręsewicz A (2007) Preconditioning
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xanthine oxidase in post-ischemic heart
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Cardiology 42(2) 400-410 DOI
101016jyjmcc200610014
7 Fabian R H Perez-Polo J R amp Kent T
A (2008) Perivascular nitric oxide and
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Heart and Circulatory Physiology 295(4)
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101152ajpheart003012007
8 Frangogiannis N G Lindsey M L
Michael L H Youker K A Bressler R
B Mendoza L H amp Entman M L
(1998) Resident cardiac mast cells
degranulate and release preformed TNF-α
initiating the cytokine cascade in
experimental canine myocardial
ischemiareperfusion Circulation 98(7)
699-710 DOI 10116101CIR987699
9 Nolly M B Caldiz C I Yeves A M
Villa-Abrille M C Morgan P E
Mondaca N A amp Ennis I L (2014)
The signaling pathway for aldosterone-
induced mitochondrial production of
superoxide anion in the myocardium
Journal of Molecular and Cellular
Cardiology 67 60-68 DOI
101016jyjmcc201312004
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10 Kleniewska P Piechota A Skibska B amp
Gorąca A (2012) The NADPH oxidase
family and its inhibitors Archivum
immunologiae et therapiae experimentalis
60(4) 277-294 DOI 101007s00005-012-
0176-z
11 Laurindo F R Araujo T L amp Abrahao T
B (2014) Nox NADPH oxidases and the
endoplasmic reticulum Antioxidants amp
Redox Signaling 20(17) 2755-2775 DOI
101089ars20135605
12 Li J M amp Shah A M (2003) Mechanism
of Endothelial Cell NADPH Oxidase
Activation by Angiotensin II ROLE OF THE
p47 phox SUBUNIT Journal of Biological
Chemistry 278(14) 12094-12100 DOI
101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
attenuated neurohumoral excitation in heart
failure a role for superoxide and nitric oxide
Basic Research in Cardiology 106(2) 273-
286 DOI 101007s00395-010-0146-8
14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 226
phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
Biochemistry 46(51) 14771-14781 DOI
101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
Immunology 33(5) 1328-1333 DOI
101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
inhibition attenuates myocardial infarction
induced heart failure and is associated with a
reduction of fibrosis and pro‐inflammatory
responses Journal of Cellular and
Molecular Medicine 15(8) 1769-1777 DOI
101111j1582-4934201001174x
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 212
decrease infarct size in myocardial IR and will
attenuate leukocyte-endothelial interactions in rat
mesenteric venules Moreover we predict that the
reduction in infarct size will parallel an
improvement in post reperfusion cardiac function
2 Methods
21 Animals
All animal procedures were approved by the
Institutional Animal Care and Use Committee at
PCOM for care and use of animals Male Sprague
Dawley (SD) rats (275-325 g Charles River
Springfield MA) were used for ex vivo and in
vivo experiments and 350-400 g male SD rats
were used for SO release assays listed below
22 Measurement of SO Release from Rat
PMNs
PMNs were isolated from male SD rats and
SO release was measured spectrophotometrically
(model 260 Gilford Nova Biotech El Cajon CA)
by the reduction of ferricytochrome c [23] The
PMNs (5x106) were re-suspended in 450 μl PBS
and incubated with ferricytochrome c (100 microM
Sigma Chemical) in a total volume of 900 μl PBS
in the presence or absence of varying
concentrations of Nox2ds-tat peptide inhibitor
([H]- R-K-K-R-R-Q-R-R-R-C-S-T-R-R-R-Q-Q-
L-NH2 MW=2452 gmol Genemed Synthesis
San Antoino TX) 10 M 40 M or 80 μM
respectively for 15 min at 37degC in
spectrophotometric cells The PMNs were
stimulated with 100 nM PMA (MW= 6168
gmol Calbiochem) in a final reaction volume of
10 ml Absorbance at 550 nm was measured
every 30 seconds for up to 360 seconds and the
change in absorbance (SO release) from PMNs
was determined relative to time 0 The dose-
response curve of Nox2ds-tat on PMN SO release
was used to determine a dose range that may be
effective in myocardial IR experiments
23 Isolated Rat Heart Preparation
Male SD rats were anesthetized
intraperitoneally (IP) with 60 mgkg pentobarbital
sodium and anticoagulated with 1000 U of
sodium heparin IP Plasma was prepared from
blood isolated from the abdominal aorta of the
same rat from which the heart was isolated from
for each cardiac perfusion experiment The
plasma was used as the vehicle for infusion of
Nox2ds-tat The hearts were rapidly excised and
perfused via the Langendorff heart preparation
[24] The aorta was cannulated and retrograde
perfused with modified Krebsrsquo buffer containing
170 mM dextrose 1200 mM NaCl 250 mM
NaHCO3 25 mM CaCl2 05 mM EDTA 59
mM KCl and 12 mM MgCl2 The modified
Krebsrsquo buffer was maintained at 37oC 80 mmHg
constant pressure aerated with 95 O2-5 CO2
and equilibrated pH between 73-74 Hearts were
immersed in an H2O jacket reservoir containing
160 ml of modified Krebsrsquo buffer at 37oC to
provide the preload The preload was established
by the volume of modified Krebsrsquo buffer that
entered the left ventricle The pressure transducer
catheter was inserted into the left ventricle of the
heart through the mitral valve Hearts were
subjected to 15 min Krebsrsquo buffer perfusion for
stabilization in order to record an initial baseline
then 30 min of global ischemia by stopping
perfusion followed by a 45 min reperfusion
period A volume of 5 ml of plasma (control) or
plasma containing Nox2ds-tat (5 μM 10 μM 40
μM or 80 μM) was injected during the first 5 min
of reperfusion by a side arm line proximal to the
heart inflow cannula at a rate of 1 mlmin All
cardiac function including coronary flow left
ventricular developed pressure ([LVDP] which is
the left ventricular end-systolic pressure [LVESP]
minus left ventricular end-diastolic pressure
[LVEDP]) maximal and minimal rate of LVDP
(+dPdtmax and ndashdPdtmin) and heart rate readings
(BPM) were taken every 5 min from initial
baseline and then throughout reperfusion
Coronary flow (mlmin) was recorded using an
inline flow meter (T106 Transonic Systems Inc
Ithaca NY) and LVDP LVESP LVEDP
+dPdtmax ndashdPdtmin and BPM readings were
recorded using a pressure transducer (SPR-524
Millar Instruments Inc Houston TX) Data was
recorded using a Powerlab Station acquisition
system (ADInstruments Grand Junction CO) At
the end of the experiment the left ventricle was
isolated and cross sectioned into 2mm pieces
from apex to base and was subjected to 1
triphenyltetrazolium chloride (TTC) staining for
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 213
15 min at 37oC to detect infarct size as previously
described [24]
24 Hindlimb IR in vivo procedure
We measured blood H2O2NO release in real-
time from sensors placed in the femoral veins of
male SD rats anesthetized with sodium
pentobarbital (60 mgkg IP for induction and 30
mgkg IP for maintenance) Thereafter one limb
was subjected to IR while the other limb was
used as a non-ischemic sham control in the same
rat The calibrated H2O2 or NO microsensors
(100 microm World Precision Instruments [WPI] Inc
Sarasota FL) were connected to a free radical
analyzer (Apollo 4000 WPI Inc) and inserted
into a 24 gauge catheter placed inside each
femoral vein as previously described by Chen et
al [25] and reviewed by Young et al [26]
Ischemia in one hindlimb was induced by
clamping the femoral arteryvein for 30 min
followed by 45 min of reperfusion Nox2ds-tat
(41 mgkg prepared in saline ~20 μM in blood)
or saline (non-drug control group) was
administered at the beginning of reperfusion as a
bolus via the jugular vein We continuously
recorded and collected the H2O2 or NO release
data at 5 min intervals during a 15 min baseline
30 min ischemia and 45 min reperfusion period
The changes in H2O2 or NO release (in pA)
during IR are expressed as relative change to
initial readings Thereafter the values were
converted to the concentration of H2O2 (μM) or
NO (nM) after correction to the respective
calibration curves The values recorded from the
femoral vein of the IR limb were subtracted from
the values of the sham limb to yield a net
difference and were recorded every 5 min
continuously on time course graphs [25]
25 Intravital Microscopy
Male SD rats were anesthetized with 60
mgkg pentobarbital sodium IP and maintained
with 30 mgkg pentobarbital IP The left carotid
artery was isolated and a PE-50 polyethylene
catheter was inserted in the left carotid artery for
monitoring of the mean arterial blood pressure
(MABP) via BP-1 Pressure Monitor (WPI Inc
Sarasota FL) After abdominal laparotomy a
loop of ileal mesentery was exteriorized and
placed in a temperature controlled Plexiglas
chamber (37 degC) for adequate superfusion of the
test solutions [27] The mesentery was placed
over a Plexiglas pedestal in the observation
chamber for visualization under a Nikon Eclipse
microscope (Nikon Co Japan) The
microcirculation was recorded with Image Pro
MDA (Media Cybernetics Bethesda MD) and
leukocyte-endothelial interactions were analyzed
offline
The rats were allowed to stabilize for 30 min
with superfusion of Krebsrsquo buffer after surgery
After stabilization a non-branched postcapillary
venule was chosen for observation An initial
recording was made to establish basal values for
leukocyte rolling adherence and transmigration
The mesentery then was superfused with the
experimental test solutions for 120 min Test
solutions included Krebsrsquo buffer alone 50 M L-
NAME alone and 50 M L-NAME in the
presence of Nox2ds-tat (5 M or 20 M) All
drugs were dissolved into the Krebsrsquo buffer The
solution was aerated with 95 N2-5 CO2 and
equilibrated at a pH of 73 to 74 at 37 degC Two
min video recordings were made at baseline 30
min 60 min 90 min and 120 min after
superfusion of the test solutions for quantification
of leukocyte rolling adherence and
transmigration [24 27]
26 Hematoxylin and Eosin (HampE) Staining After the experiment the loop of the ileal
mesentery that was superfused during the
experiment was removed and quickly stored in 4
paraformaldehyde for histological analysis of
leukocyte adherence and transmigration by HampE
staining Three representative sections of the ileal
mesentery from Krebsrsquo buffer 50 microM L-NAME
and 50 microM L-NAME in the presence of Nox2ds-
tat (5 M or 20 microM) were selected for
histological analysis Tissue samples were
selected from experiments representative of the
group mean of intravital microscopy (Table 2)
The tissue was embedded in paraffin and
sectioned into 45 microm serial sections and placed
onto glass slides Sections were deparaffinized
and rehydrated then stained with HampE [27]
Under light microscopy leukocyte adherence and
transmigration was counted in areas containing
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 214
venulesarterioles within the serosa and the
mesentery and expressed as adhered and
transmigrated leukocytesmm2 in tissue
27 Statistical Analysis
All data in the text and figures are presented
as means plusmn standard error of the mean (SEM)
The data was analyzed by analysis of variance
(ANOVA) using post hoc analysis with the
Student-Newman-Keuls test for all experiments
except for hindlimb IR in which a studentrsquos t-test
was used A p value of lt005 was considered to
be statistically significant
3 Results
31 PMN SO release
PMA is a broad-spectrum activator of protein
kinase C (PKC) which is essential in promoting
NADPH oxidase assembly at the PMN
membrane to generate SO release [28]We found
that Nox2ds-tat exhibited a dose-dependent
reduction in PMA-stimulated PMN SO release
(Figure 3) Doses of Nox2ds-tat (10 μM and 40
μM) did not significantly decrease PMN SO
release whereas a dose of 80 μM significantly
inhibited PMA-induced SO release by 37 plusmn 7
(plt005 compared to PMA alone) The data
generated from this assay was used to test a
similar dose range of Nox2ds-tat in isolated rat
heart IR experiments
Figure 3 PMA (100 nM) induced SO release in PMNs peak response read at 360s post stimulation Nox2ds-tat dose
dependently inhibited SO release from PMNs by 37 plusmn 7 (80 microM n=4) when compared to PMNs treated with PMA
alone (plt005 vs PMA control)
32 Cardiac function and infarct size
Nox2ds-tat given at reperfusion dose-
dependently attenuated IR induced cardiac
contractile dysfunction and recovered post-
reperfused LVDP to 47 plusmn 7 (5 μM n=7) 69 plusmn
13 (10 μM n=6 plt005) 68 plusmn 7 (40 μM
n=6 plt005) and 77 plusmn 7 (80 μM n=6 plt005)
of initial LVDP at 45 min post-reperfusion when
compared to control IR hearts that only
recovered to 46 plusmn 6 (n=14) (Table 1) Initial
baseline values for cardiac function parameters
did not differ significantly among groups
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 215
Table 1 Cardiac function parameters among different experimental groups
Cardiac Parameters Control IR
(n=14)
IR +
Nox2ds-tat 5
μM
(n=7)
IR +
Nox2ds-tat
10 μM
(n=6)
IR +
Nox2ds-tat
40 μM
(n=6)
IR +
Nox2ds-tat
80 μM
(n=6)
Initial LVDP (mmHg) 87plusmn35 91plusmn31 89plusmn34 92plusmn39 88plusmn33
Final LVDP (mmHg) 40plusmn50 43plusmn66 61plusmn121 63plusmn62 68plusmn62
Initial LVESP (mmHg) 99plusmn38 101plusmn39 105plusmn48 103plusmn46 101plusmn37
Final LVESP (mmHg) 100plusmn53 108plusmn57 107plusmn119 110plusmn75 124plusmn97
Initial LVEDP (mmHg) 11plusmn10 94plusmn13 16plusmn20 11plusmn13 13plusmn07
Final LVEDP(mmHg) 61plusmn51 64plusmn42 46plusmn61 47plusmn75
56plusmn56
Initial +dPdtmax (mmHgsec) 2259plusmn71 2335plusmn61 2333plusmn66 2346plusmn77 2277plusmn79
Final +dPdtmax (mmHgsec) 894plusmn105 832plusmn124 1285plusmn232 1258plusmn120
1413plusmn100
Initial -dPdtmin (mmHgsec) -1510plusmn67 -1616plusmn98 -1607plusmn84 -1550plusmn124 -1524plusmn106
Final -dPdtmin (mmHgsec) -704plusmn65 -660plusmn136 -879plusmn187 -939plusmn108 -1116plusmn146
Initial coronary flow
(mlmin) 162plusmn12 144plusmn064 181plusmn14 180plusmn25 152plusmn10
Final coronary flow (mlmin) 79plusmn064 79plusmn070 92plusmn15 93plusmn17 95plusmn15
Initial Heart Rate (BPM) 271plusmn128 277plusmn123 280plusmn78 272plusmn88 265plusmn32
Final Heart Rate (BPM) 246plusmn188 234plusmn199 230plusmn105 249plusmn136 237plusmn119
plt005 vs control IR final cardiac function parameters plt005
plt001 vs Nox2ds-tat 5μM
Similarly Nox2ds-tat reduced infarct
sizearea-at-risk when compared to the IR
control group (Figure 4 Panel A-B) The cross
section for the IR control group shows the
majority of the infarct in the mid-wall area
whereas the Nox2ds-tat treated IR hearts showed
less infarct area (Figure 4 Panel A) Figure 4
Panel B shows the ratio of this tissue at risk
When compared to the IR control group (46 plusmn
2) there was a significant decrease in total
infarct size in hearts treated with Nox2ds-tat 30 plusmn
4 (5 μM) 15 plusmn 14 (10 μM) 23 plusmn 2 (40
microM) and 19 plusmn 16 (80 microM)(plt001 for all
groups) Moreover the 10 μM and 80 μM doses
showed significantly less infarct size compared to
the 5 μM dose (plt001 and plt005 respectively)
suggesting a dose-dependent effect of Nox2ds-tat
on infarct size (Figure 4 Panel A-B)
Other cardiac function parameters are
illustrated in Table 1 Of interest Nox2ds-tat
dose-dependently recovered post-reperfused final
+dPdtmax by 36 plusmn 53 (5 μM) 55 plusmn10 (10
μM) 54 plusmn 4 (40 microM plt005) 62 plusmn 4 (80 microM
plt005) at 45 min reperfusion compared to
control IR hearts that only recovered to 40 plusmn 5
Sham hearts (n=6) were not subjected to IR and
maintained cardiac function parameters and flow
by gt90 and had lt5 infarct sizearea-at- risk
throughout the experimental time-course (data
not shown)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 216
Figure 4 Panel A Representative images of TTC staining and percentage of left ventricular heart tissue at risk among
different experimental groups Viable tissue stains red infarcted tissue did not retain TTC staining Nox2ds-tat dose
dependently attenuated infarct size Panel B Ratio of left ventricular heart tissue at risk for myocardial dysfunction
out of total tissue weight as determined by TTC staining of representative heart sections When compared to the IR
control group which had an infarct size of 46plusmn2 there was a significant decrease in total infarct size in heart sections
treated with Nox2ds-tat Nox2ds-tat dose-dependently attenuated infarct sizes to 30 plusmn 4 (5 μM n=7) 15 plusmn 14 (10
μM n=6) 23 plusmn 2 (40 microM n=6) and 19 plusmn 16 (80 microM n=6) (plt001 vs control tissue n=14) There was also a
significant decrease in infarct size observed in 10 μM (plt001) and 80 microM (plt005) compared to 5 μM Nox2ds-tat
(plt001 vs control IR plt005 vs Nox2ds-tat 5 microM plt001 vs Nox2ds-tat 5microM)
33 Hindlimb IR in vivo
331 Nox2ds-tat reduced blood H2O2 levels
The blood H2O2 values in the IR hindlimb of
saline controls significantly increased to 17 plusmn 03
microM (15 min post-reperfusion) above sham values
Thereafter a relative difference of 12-14 μM
between the IR and sham limbs was maintained
throughout the remainder of reperfusion (Figure 5
Panel A) By contrast Nox2ds-tat (41 mgkg
~20 μM in blood) treated animals in the IR
hindlimb did not significantly differ from the
sham hindlimb throughout reperfusion (Figure 5
Panel B) The relative difference in blood H2O2
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant reduction in the IR induced effect on
blood H2O2 by 143 plusmn 02 μM at the end of
reperfusion (Figure 5 Panel C)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 217
Figure 5 Panel A Relative difference in measured blood H2O2 between IR and sham femoral veins in saline controls
The saline IR vein showed a significant increase in blood H2O2 by 17 plusmn 03 microM (5 min) relative to the sham femoral
vein This increase above sham femoral vein values was maintained throughout reperfusion stabilizing at 14 plusmn 02 microM
(45 min) (plt005 plt001 vs sham IR femoral vein) Panel B Relative difference in measured blood H2O2
between IR and sham femoral veins in Nox2ds-tat (41 mgkg ~20 microM) treated rats The IR femoral vein treated with
Nox2ds-tat showed a transient increase in H2O2 when compared to the sham femoral vein However this difference
between the two limbs decreased throughout the remainder of the experiment ending at ~0 microM The measured values
in the two groups were not statistically different at any point in the experiment Panel C Relative difference in
measured blood H2O2 betweenNox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline
values) Nox2ds-tat infusion at reperfusion significantly decreased the blood H2O2 concentration when compared to
rats infused with saline At the end of the 45 min reperfusion period there was a significant reduction of 14 microM
between blood H2O2 measured in Nox2ds-tat 20 microM treated rats relative to saline control (plt005 plt001 vs
saline treated femoral veins)
B
A
C
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 218
A
B
C
Figure 6 Panel A Relative difference in measured blood NO between IR and sham femoral veins in saline controls
Overall the IR femoral vein showed a significant decrease in NO concentration stabilizing at a difference of 90 plusmn 21
nM when compared to the sham femoral vein at the end of the 45 min reperfusion period (plt005 plt001 vs sham
IR femoral vein) Panel B Relative difference in measured blood NO between IR and sham femoral veins in Nox2ds-
tat (41 mgkg ~20 microM) treated rats In Nox2ds-tat treated animals the IR femoral vein resulted in 37 plusmn 12 nM higher
blood NO concentration than the sham limb at the end of the 45 min reperfusion period however this difference was
not statistically significant (plt005 plt001 vs sham IR femoral vein) Panel C Relative difference in measured
blood NO in Nox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline values) Nox2ds-tat
given at reperfusion significantly increased the total blood NO concentration when compared to saline controls At the
end of the 45 min reperfusion period there was a significant increase of 127 nM in blood NO in Nox2ds-tat treated
animals relative to saline controls (plt005 plt001 vs saline treated femoral veins)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 219
332 Nox2ds-tat prevented the decrease in
blood NO levels
In saline controls blood NO levels steadily
decreased in the IR limb throughout the
reperfusion period leading to significantly lower
blood NO bioavailability (90 nM below sham) as
early as 20 min into the reperfusion period
(Figure 6 Panel A) By contrast blood NO in the
IR hindlimb of Nox2ds-tat (41 mgkg ~20 μM
in blood) treated animals did not significantly
differ from sham hindlimb values during the
reperfusion period While a linear trend close to
sham values was observed the Nox2ds-tat treated
grouprsquos blood NO levels did rise to 37 nM above
sham values at the end of reperfusion (Figure 6
Panel B) The relative difference in blood NO
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant increase in the IR induced effect on
NO release by 127 nM at the end of reperfusion
(Figure 6 Panel C)
34 L-NAME induced Leukocyte endothelial
interactions
341 Intravital Microscopy
There was no significant difference among
initial baseline values in leukocyte rolling
adherence and transmigration among all study
groups In all parameters the Krebsrsquo buffer group
did not significantly change from baseline L-
NAME (50 microM) significantly increased leukocyte
rolling adherence and transmigration when
compared to Krebsrsquo at T=120 min by 82 plusmn 11
(plt001) 22 plusmn 5 (plt001) and 21 plusmn 4 (plt001)
respectively (Table 2) Nox2ds-tat (5 microM) did not
significantly attenuate L-NAME induced
leukocyte-endothelial interactions except for final
transmigration (plt005) By contrast Nox2ds-tat
(20 microM) significantly decreased the L-NAME
induced effect by 3-5 fold on all parameters
(plt001) (Table 2)
Table 2 The comparision of initial and final (120 min of reperfusion) leukocyte rolling adherence and transmigration
between Krebsrsquo L-NAME and L-NAME + Nox2ds-tat (5 μM 20 μM) groups in rat mesenteric venules
Intravital microscopy Parameters
Krebsrsquo
(n=6)
L-NAME
(n=5)
L-NAME +
Nox2ds-tat 5 μM
(n=4)
L-NAME +
Nox2ds-tat 20 μM
(n=6)
Initial Rolling (numbermin) 8plusmn3 11plusmn3 13plusmn3 8plusmn2
Final Rolling (numbermin) 16plusmn5 82plusmn11
72plusmn13 16plusmn6++
Initial Adherence (number100um) 2plusmn2 1plusmn0 3plusmn0 1plusmn1
Final Adherence (number100um) 4plusmn2 22plusmn5 17plusmn1 8plusmn4
Initial Transmigration (number) 1plusmn0 1plusmn0 1plusmn0 1plusmn0
Final Transmigration (number) 1plusmn0 21plusmn4 9plusmn1
4plusmn2
Plt005 Plt001 compared to Krebsrsquo groupPlt005
Plt001 compared to L-NAME
++Plt001 compared to L-
NAME + Nox2ds-tat 5 μM
342 HampE Staining
The Krebsrsquocontrol group exhibited only 73 plusmn
33 and 62 plusmn 30 leukocytesmm2 of mesenteric
tissue These values represent basal numbers of
leukocyte adherence and transmigration in this
tissue bed respectively L-NAME treatment
exhibited a marked increase (~4-fold) in
leukocyte vascular adherence and transmigration
compared to Krebsrsquo buffer (299 plusmn 23 and 271 plusmn
26 leukocytesmm2 of tissue respectively plt001
for both values) Nox2ds-tat dose-dependently
attenuated the L-NAME induced effect on
leukocyte adherence (199 plusmn 13 for 5 microM at
plt005 and 83 plusmn 9 for 20 microM plt001) and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
References
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Myocardial reperfusion injury New England
Journal of Medicine 357(11) 1121-1135
DOI 101056NEJMra071667
2 Hausenloy D J amp Yellon D M (2013)
Myocardial ischemia-reperfusion injury a
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Clinical Investigation 123(1) 92-100 DOI
101172JCI62874
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6363(96)00073-9
4 Ago T Kuroda J Kamouchi M
Sadoshima J amp Kitazono T (2011)
Pathophysiological Roles of NADPH
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system-review and perspective Circulation
Journal 75(8) 1791-1800 DOI
101253circjCJ-11-0388
5 Doughan A K Harrison D G amp Dikalov
S I (2008) Molecular Mechanisms of
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Oxidative Damage and Vascular Endothelial
Dysfunction Circulation research 102(4)
488-496 DOI
101161CIRCRESAHA107162800
6 Duda M Konior A Klemenska E amp
Beręsewicz A (2007) Preconditioning
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induced activation of NADPH oxidase and
xanthine oxidase in post-ischemic heart
Journal of Molecular and Cellular
Cardiology 42(2) 400-410 DOI
101016jyjmcc200610014
7 Fabian R H Perez-Polo J R amp Kent T
A (2008) Perivascular nitric oxide and
superoxide in neonatal cerebral hypoxia-
ischemia American Journal of Physiology-
Heart and Circulatory Physiology 295(4)
H1809-H1814 DOI
101152ajpheart003012007
8 Frangogiannis N G Lindsey M L
Michael L H Youker K A Bressler R
B Mendoza L H amp Entman M L
(1998) Resident cardiac mast cells
degranulate and release preformed TNF-α
initiating the cytokine cascade in
experimental canine myocardial
ischemiareperfusion Circulation 98(7)
699-710 DOI 10116101CIR987699
9 Nolly M B Caldiz C I Yeves A M
Villa-Abrille M C Morgan P E
Mondaca N A amp Ennis I L (2014)
The signaling pathway for aldosterone-
induced mitochondrial production of
superoxide anion in the myocardium
Journal of Molecular and Cellular
Cardiology 67 60-68 DOI
101016jyjmcc201312004
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 225
10 Kleniewska P Piechota A Skibska B amp
Gorąca A (2012) The NADPH oxidase
family and its inhibitors Archivum
immunologiae et therapiae experimentalis
60(4) 277-294 DOI 101007s00005-012-
0176-z
11 Laurindo F R Araujo T L amp Abrahao T
B (2014) Nox NADPH oxidases and the
endoplasmic reticulum Antioxidants amp
Redox Signaling 20(17) 2755-2775 DOI
101089ars20135605
12 Li J M amp Shah A M (2003) Mechanism
of Endothelial Cell NADPH Oxidase
Activation by Angiotensin II ROLE OF THE
p47 phox SUBUNIT Journal of Biological
Chemistry 278(14) 12094-12100 DOI
101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
attenuated neurohumoral excitation in heart
failure a role for superoxide and nitric oxide
Basic Research in Cardiology 106(2) 273-
286 DOI 101007s00395-010-0146-8
14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 226
phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
Biochemistry 46(51) 14771-14781 DOI
101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
Immunology 33(5) 1328-1333 DOI
101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
inhibition attenuates myocardial infarction
induced heart failure and is associated with a
reduction of fibrosis and pro‐inflammatory
responses Journal of Cellular and
Molecular Medicine 15(8) 1769-1777 DOI
101111j1582-4934201001174x
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 213
15 min at 37oC to detect infarct size as previously
described [24]
24 Hindlimb IR in vivo procedure
We measured blood H2O2NO release in real-
time from sensors placed in the femoral veins of
male SD rats anesthetized with sodium
pentobarbital (60 mgkg IP for induction and 30
mgkg IP for maintenance) Thereafter one limb
was subjected to IR while the other limb was
used as a non-ischemic sham control in the same
rat The calibrated H2O2 or NO microsensors
(100 microm World Precision Instruments [WPI] Inc
Sarasota FL) were connected to a free radical
analyzer (Apollo 4000 WPI Inc) and inserted
into a 24 gauge catheter placed inside each
femoral vein as previously described by Chen et
al [25] and reviewed by Young et al [26]
Ischemia in one hindlimb was induced by
clamping the femoral arteryvein for 30 min
followed by 45 min of reperfusion Nox2ds-tat
(41 mgkg prepared in saline ~20 μM in blood)
or saline (non-drug control group) was
administered at the beginning of reperfusion as a
bolus via the jugular vein We continuously
recorded and collected the H2O2 or NO release
data at 5 min intervals during a 15 min baseline
30 min ischemia and 45 min reperfusion period
The changes in H2O2 or NO release (in pA)
during IR are expressed as relative change to
initial readings Thereafter the values were
converted to the concentration of H2O2 (μM) or
NO (nM) after correction to the respective
calibration curves The values recorded from the
femoral vein of the IR limb were subtracted from
the values of the sham limb to yield a net
difference and were recorded every 5 min
continuously on time course graphs [25]
25 Intravital Microscopy
Male SD rats were anesthetized with 60
mgkg pentobarbital sodium IP and maintained
with 30 mgkg pentobarbital IP The left carotid
artery was isolated and a PE-50 polyethylene
catheter was inserted in the left carotid artery for
monitoring of the mean arterial blood pressure
(MABP) via BP-1 Pressure Monitor (WPI Inc
Sarasota FL) After abdominal laparotomy a
loop of ileal mesentery was exteriorized and
placed in a temperature controlled Plexiglas
chamber (37 degC) for adequate superfusion of the
test solutions [27] The mesentery was placed
over a Plexiglas pedestal in the observation
chamber for visualization under a Nikon Eclipse
microscope (Nikon Co Japan) The
microcirculation was recorded with Image Pro
MDA (Media Cybernetics Bethesda MD) and
leukocyte-endothelial interactions were analyzed
offline
The rats were allowed to stabilize for 30 min
with superfusion of Krebsrsquo buffer after surgery
After stabilization a non-branched postcapillary
venule was chosen for observation An initial
recording was made to establish basal values for
leukocyte rolling adherence and transmigration
The mesentery then was superfused with the
experimental test solutions for 120 min Test
solutions included Krebsrsquo buffer alone 50 M L-
NAME alone and 50 M L-NAME in the
presence of Nox2ds-tat (5 M or 20 M) All
drugs were dissolved into the Krebsrsquo buffer The
solution was aerated with 95 N2-5 CO2 and
equilibrated at a pH of 73 to 74 at 37 degC Two
min video recordings were made at baseline 30
min 60 min 90 min and 120 min after
superfusion of the test solutions for quantification
of leukocyte rolling adherence and
transmigration [24 27]
26 Hematoxylin and Eosin (HampE) Staining After the experiment the loop of the ileal
mesentery that was superfused during the
experiment was removed and quickly stored in 4
paraformaldehyde for histological analysis of
leukocyte adherence and transmigration by HampE
staining Three representative sections of the ileal
mesentery from Krebsrsquo buffer 50 microM L-NAME
and 50 microM L-NAME in the presence of Nox2ds-
tat (5 M or 20 microM) were selected for
histological analysis Tissue samples were
selected from experiments representative of the
group mean of intravital microscopy (Table 2)
The tissue was embedded in paraffin and
sectioned into 45 microm serial sections and placed
onto glass slides Sections were deparaffinized
and rehydrated then stained with HampE [27]
Under light microscopy leukocyte adherence and
transmigration was counted in areas containing
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 214
venulesarterioles within the serosa and the
mesentery and expressed as adhered and
transmigrated leukocytesmm2 in tissue
27 Statistical Analysis
All data in the text and figures are presented
as means plusmn standard error of the mean (SEM)
The data was analyzed by analysis of variance
(ANOVA) using post hoc analysis with the
Student-Newman-Keuls test for all experiments
except for hindlimb IR in which a studentrsquos t-test
was used A p value of lt005 was considered to
be statistically significant
3 Results
31 PMN SO release
PMA is a broad-spectrum activator of protein
kinase C (PKC) which is essential in promoting
NADPH oxidase assembly at the PMN
membrane to generate SO release [28]We found
that Nox2ds-tat exhibited a dose-dependent
reduction in PMA-stimulated PMN SO release
(Figure 3) Doses of Nox2ds-tat (10 μM and 40
μM) did not significantly decrease PMN SO
release whereas a dose of 80 μM significantly
inhibited PMA-induced SO release by 37 plusmn 7
(plt005 compared to PMA alone) The data
generated from this assay was used to test a
similar dose range of Nox2ds-tat in isolated rat
heart IR experiments
Figure 3 PMA (100 nM) induced SO release in PMNs peak response read at 360s post stimulation Nox2ds-tat dose
dependently inhibited SO release from PMNs by 37 plusmn 7 (80 microM n=4) when compared to PMNs treated with PMA
alone (plt005 vs PMA control)
32 Cardiac function and infarct size
Nox2ds-tat given at reperfusion dose-
dependently attenuated IR induced cardiac
contractile dysfunction and recovered post-
reperfused LVDP to 47 plusmn 7 (5 μM n=7) 69 plusmn
13 (10 μM n=6 plt005) 68 plusmn 7 (40 μM
n=6 plt005) and 77 plusmn 7 (80 μM n=6 plt005)
of initial LVDP at 45 min post-reperfusion when
compared to control IR hearts that only
recovered to 46 plusmn 6 (n=14) (Table 1) Initial
baseline values for cardiac function parameters
did not differ significantly among groups
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 215
Table 1 Cardiac function parameters among different experimental groups
Cardiac Parameters Control IR
(n=14)
IR +
Nox2ds-tat 5
μM
(n=7)
IR +
Nox2ds-tat
10 μM
(n=6)
IR +
Nox2ds-tat
40 μM
(n=6)
IR +
Nox2ds-tat
80 μM
(n=6)
Initial LVDP (mmHg) 87plusmn35 91plusmn31 89plusmn34 92plusmn39 88plusmn33
Final LVDP (mmHg) 40plusmn50 43plusmn66 61plusmn121 63plusmn62 68plusmn62
Initial LVESP (mmHg) 99plusmn38 101plusmn39 105plusmn48 103plusmn46 101plusmn37
Final LVESP (mmHg) 100plusmn53 108plusmn57 107plusmn119 110plusmn75 124plusmn97
Initial LVEDP (mmHg) 11plusmn10 94plusmn13 16plusmn20 11plusmn13 13plusmn07
Final LVEDP(mmHg) 61plusmn51 64plusmn42 46plusmn61 47plusmn75
56plusmn56
Initial +dPdtmax (mmHgsec) 2259plusmn71 2335plusmn61 2333plusmn66 2346plusmn77 2277plusmn79
Final +dPdtmax (mmHgsec) 894plusmn105 832plusmn124 1285plusmn232 1258plusmn120
1413plusmn100
Initial -dPdtmin (mmHgsec) -1510plusmn67 -1616plusmn98 -1607plusmn84 -1550plusmn124 -1524plusmn106
Final -dPdtmin (mmHgsec) -704plusmn65 -660plusmn136 -879plusmn187 -939plusmn108 -1116plusmn146
Initial coronary flow
(mlmin) 162plusmn12 144plusmn064 181plusmn14 180plusmn25 152plusmn10
Final coronary flow (mlmin) 79plusmn064 79plusmn070 92plusmn15 93plusmn17 95plusmn15
Initial Heart Rate (BPM) 271plusmn128 277plusmn123 280plusmn78 272plusmn88 265plusmn32
Final Heart Rate (BPM) 246plusmn188 234plusmn199 230plusmn105 249plusmn136 237plusmn119
plt005 vs control IR final cardiac function parameters plt005
plt001 vs Nox2ds-tat 5μM
Similarly Nox2ds-tat reduced infarct
sizearea-at-risk when compared to the IR
control group (Figure 4 Panel A-B) The cross
section for the IR control group shows the
majority of the infarct in the mid-wall area
whereas the Nox2ds-tat treated IR hearts showed
less infarct area (Figure 4 Panel A) Figure 4
Panel B shows the ratio of this tissue at risk
When compared to the IR control group (46 plusmn
2) there was a significant decrease in total
infarct size in hearts treated with Nox2ds-tat 30 plusmn
4 (5 μM) 15 plusmn 14 (10 μM) 23 plusmn 2 (40
microM) and 19 plusmn 16 (80 microM)(plt001 for all
groups) Moreover the 10 μM and 80 μM doses
showed significantly less infarct size compared to
the 5 μM dose (plt001 and plt005 respectively)
suggesting a dose-dependent effect of Nox2ds-tat
on infarct size (Figure 4 Panel A-B)
Other cardiac function parameters are
illustrated in Table 1 Of interest Nox2ds-tat
dose-dependently recovered post-reperfused final
+dPdtmax by 36 plusmn 53 (5 μM) 55 plusmn10 (10
μM) 54 plusmn 4 (40 microM plt005) 62 plusmn 4 (80 microM
plt005) at 45 min reperfusion compared to
control IR hearts that only recovered to 40 plusmn 5
Sham hearts (n=6) were not subjected to IR and
maintained cardiac function parameters and flow
by gt90 and had lt5 infarct sizearea-at- risk
throughout the experimental time-course (data
not shown)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 216
Figure 4 Panel A Representative images of TTC staining and percentage of left ventricular heart tissue at risk among
different experimental groups Viable tissue stains red infarcted tissue did not retain TTC staining Nox2ds-tat dose
dependently attenuated infarct size Panel B Ratio of left ventricular heart tissue at risk for myocardial dysfunction
out of total tissue weight as determined by TTC staining of representative heart sections When compared to the IR
control group which had an infarct size of 46plusmn2 there was a significant decrease in total infarct size in heart sections
treated with Nox2ds-tat Nox2ds-tat dose-dependently attenuated infarct sizes to 30 plusmn 4 (5 μM n=7) 15 plusmn 14 (10
μM n=6) 23 plusmn 2 (40 microM n=6) and 19 plusmn 16 (80 microM n=6) (plt001 vs control tissue n=14) There was also a
significant decrease in infarct size observed in 10 μM (plt001) and 80 microM (plt005) compared to 5 μM Nox2ds-tat
(plt001 vs control IR plt005 vs Nox2ds-tat 5 microM plt001 vs Nox2ds-tat 5microM)
33 Hindlimb IR in vivo
331 Nox2ds-tat reduced blood H2O2 levels
The blood H2O2 values in the IR hindlimb of
saline controls significantly increased to 17 plusmn 03
microM (15 min post-reperfusion) above sham values
Thereafter a relative difference of 12-14 μM
between the IR and sham limbs was maintained
throughout the remainder of reperfusion (Figure 5
Panel A) By contrast Nox2ds-tat (41 mgkg
~20 μM in blood) treated animals in the IR
hindlimb did not significantly differ from the
sham hindlimb throughout reperfusion (Figure 5
Panel B) The relative difference in blood H2O2
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant reduction in the IR induced effect on
blood H2O2 by 143 plusmn 02 μM at the end of
reperfusion (Figure 5 Panel C)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 217
Figure 5 Panel A Relative difference in measured blood H2O2 between IR and sham femoral veins in saline controls
The saline IR vein showed a significant increase in blood H2O2 by 17 plusmn 03 microM (5 min) relative to the sham femoral
vein This increase above sham femoral vein values was maintained throughout reperfusion stabilizing at 14 plusmn 02 microM
(45 min) (plt005 plt001 vs sham IR femoral vein) Panel B Relative difference in measured blood H2O2
between IR and sham femoral veins in Nox2ds-tat (41 mgkg ~20 microM) treated rats The IR femoral vein treated with
Nox2ds-tat showed a transient increase in H2O2 when compared to the sham femoral vein However this difference
between the two limbs decreased throughout the remainder of the experiment ending at ~0 microM The measured values
in the two groups were not statistically different at any point in the experiment Panel C Relative difference in
measured blood H2O2 betweenNox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline
values) Nox2ds-tat infusion at reperfusion significantly decreased the blood H2O2 concentration when compared to
rats infused with saline At the end of the 45 min reperfusion period there was a significant reduction of 14 microM
between blood H2O2 measured in Nox2ds-tat 20 microM treated rats relative to saline control (plt005 plt001 vs
saline treated femoral veins)
B
A
C
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 218
A
B
C
Figure 6 Panel A Relative difference in measured blood NO between IR and sham femoral veins in saline controls
Overall the IR femoral vein showed a significant decrease in NO concentration stabilizing at a difference of 90 plusmn 21
nM when compared to the sham femoral vein at the end of the 45 min reperfusion period (plt005 plt001 vs sham
IR femoral vein) Panel B Relative difference in measured blood NO between IR and sham femoral veins in Nox2ds-
tat (41 mgkg ~20 microM) treated rats In Nox2ds-tat treated animals the IR femoral vein resulted in 37 plusmn 12 nM higher
blood NO concentration than the sham limb at the end of the 45 min reperfusion period however this difference was
not statistically significant (plt005 plt001 vs sham IR femoral vein) Panel C Relative difference in measured
blood NO in Nox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline values) Nox2ds-tat
given at reperfusion significantly increased the total blood NO concentration when compared to saline controls At the
end of the 45 min reperfusion period there was a significant increase of 127 nM in blood NO in Nox2ds-tat treated
animals relative to saline controls (plt005 plt001 vs saline treated femoral veins)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 219
332 Nox2ds-tat prevented the decrease in
blood NO levels
In saline controls blood NO levels steadily
decreased in the IR limb throughout the
reperfusion period leading to significantly lower
blood NO bioavailability (90 nM below sham) as
early as 20 min into the reperfusion period
(Figure 6 Panel A) By contrast blood NO in the
IR hindlimb of Nox2ds-tat (41 mgkg ~20 μM
in blood) treated animals did not significantly
differ from sham hindlimb values during the
reperfusion period While a linear trend close to
sham values was observed the Nox2ds-tat treated
grouprsquos blood NO levels did rise to 37 nM above
sham values at the end of reperfusion (Figure 6
Panel B) The relative difference in blood NO
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant increase in the IR induced effect on
NO release by 127 nM at the end of reperfusion
(Figure 6 Panel C)
34 L-NAME induced Leukocyte endothelial
interactions
341 Intravital Microscopy
There was no significant difference among
initial baseline values in leukocyte rolling
adherence and transmigration among all study
groups In all parameters the Krebsrsquo buffer group
did not significantly change from baseline L-
NAME (50 microM) significantly increased leukocyte
rolling adherence and transmigration when
compared to Krebsrsquo at T=120 min by 82 plusmn 11
(plt001) 22 plusmn 5 (plt001) and 21 plusmn 4 (plt001)
respectively (Table 2) Nox2ds-tat (5 microM) did not
significantly attenuate L-NAME induced
leukocyte-endothelial interactions except for final
transmigration (plt005) By contrast Nox2ds-tat
(20 microM) significantly decreased the L-NAME
induced effect by 3-5 fold on all parameters
(plt001) (Table 2)
Table 2 The comparision of initial and final (120 min of reperfusion) leukocyte rolling adherence and transmigration
between Krebsrsquo L-NAME and L-NAME + Nox2ds-tat (5 μM 20 μM) groups in rat mesenteric venules
Intravital microscopy Parameters
Krebsrsquo
(n=6)
L-NAME
(n=5)
L-NAME +
Nox2ds-tat 5 μM
(n=4)
L-NAME +
Nox2ds-tat 20 μM
(n=6)
Initial Rolling (numbermin) 8plusmn3 11plusmn3 13plusmn3 8plusmn2
Final Rolling (numbermin) 16plusmn5 82plusmn11
72plusmn13 16plusmn6++
Initial Adherence (number100um) 2plusmn2 1plusmn0 3plusmn0 1plusmn1
Final Adherence (number100um) 4plusmn2 22plusmn5 17plusmn1 8plusmn4
Initial Transmigration (number) 1plusmn0 1plusmn0 1plusmn0 1plusmn0
Final Transmigration (number) 1plusmn0 21plusmn4 9plusmn1
4plusmn2
Plt005 Plt001 compared to Krebsrsquo groupPlt005
Plt001 compared to L-NAME
++Plt001 compared to L-
NAME + Nox2ds-tat 5 μM
342 HampE Staining
The Krebsrsquocontrol group exhibited only 73 plusmn
33 and 62 plusmn 30 leukocytesmm2 of mesenteric
tissue These values represent basal numbers of
leukocyte adherence and transmigration in this
tissue bed respectively L-NAME treatment
exhibited a marked increase (~4-fold) in
leukocyte vascular adherence and transmigration
compared to Krebsrsquo buffer (299 plusmn 23 and 271 plusmn
26 leukocytesmm2 of tissue respectively plt001
for both values) Nox2ds-tat dose-dependently
attenuated the L-NAME induced effect on
leukocyte adherence (199 plusmn 13 for 5 microM at
plt005 and 83 plusmn 9 for 20 microM plt001) and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
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Myocardial reperfusion injury New England
Journal of Medicine 357(11) 1121-1135
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2 Hausenloy D J amp Yellon D M (2013)
Myocardial ischemia-reperfusion injury a
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Clinical Investigation 123(1) 92-100 DOI
101172JCI62874
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6363(96)00073-9
4 Ago T Kuroda J Kamouchi M
Sadoshima J amp Kitazono T (2011)
Pathophysiological Roles of NADPH
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101253circjCJ-11-0388
5 Doughan A K Harrison D G amp Dikalov
S I (2008) Molecular Mechanisms of
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488-496 DOI
101161CIRCRESAHA107162800
6 Duda M Konior A Klemenska E amp
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Cardiology 42(2) 400-410 DOI
101016jyjmcc200610014
7 Fabian R H Perez-Polo J R amp Kent T
A (2008) Perivascular nitric oxide and
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101152ajpheart003012007
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Michael L H Youker K A Bressler R
B Mendoza L H amp Entman M L
(1998) Resident cardiac mast cells
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Villa-Abrille M C Morgan P E
Mondaca N A amp Ennis I L (2014)
The signaling pathway for aldosterone-
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Journal of Molecular and Cellular
Cardiology 67 60-68 DOI
101016jyjmcc201312004
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10 Kleniewska P Piechota A Skibska B amp
Gorąca A (2012) The NADPH oxidase
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60(4) 277-294 DOI 101007s00005-012-
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11 Laurindo F R Araujo T L amp Abrahao T
B (2014) Nox NADPH oxidases and the
endoplasmic reticulum Antioxidants amp
Redox Signaling 20(17) 2755-2775 DOI
101089ars20135605
12 Li J M amp Shah A M (2003) Mechanism
of Endothelial Cell NADPH Oxidase
Activation by Angiotensin II ROLE OF THE
p47 phox SUBUNIT Journal of Biological
Chemistry 278(14) 12094-12100 DOI
101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
attenuated neurohumoral excitation in heart
failure a role for superoxide and nitric oxide
Basic Research in Cardiology 106(2) 273-
286 DOI 101007s00395-010-0146-8
14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
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phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
Biochemistry 46(51) 14771-14781 DOI
101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
Immunology 33(5) 1328-1333 DOI
101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
inhibition attenuates myocardial infarction
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101111j1582-4934201001174x
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38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
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Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
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Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
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Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 214
venulesarterioles within the serosa and the
mesentery and expressed as adhered and
transmigrated leukocytesmm2 in tissue
27 Statistical Analysis
All data in the text and figures are presented
as means plusmn standard error of the mean (SEM)
The data was analyzed by analysis of variance
(ANOVA) using post hoc analysis with the
Student-Newman-Keuls test for all experiments
except for hindlimb IR in which a studentrsquos t-test
was used A p value of lt005 was considered to
be statistically significant
3 Results
31 PMN SO release
PMA is a broad-spectrum activator of protein
kinase C (PKC) which is essential in promoting
NADPH oxidase assembly at the PMN
membrane to generate SO release [28]We found
that Nox2ds-tat exhibited a dose-dependent
reduction in PMA-stimulated PMN SO release
(Figure 3) Doses of Nox2ds-tat (10 μM and 40
μM) did not significantly decrease PMN SO
release whereas a dose of 80 μM significantly
inhibited PMA-induced SO release by 37 plusmn 7
(plt005 compared to PMA alone) The data
generated from this assay was used to test a
similar dose range of Nox2ds-tat in isolated rat
heart IR experiments
Figure 3 PMA (100 nM) induced SO release in PMNs peak response read at 360s post stimulation Nox2ds-tat dose
dependently inhibited SO release from PMNs by 37 plusmn 7 (80 microM n=4) when compared to PMNs treated with PMA
alone (plt005 vs PMA control)
32 Cardiac function and infarct size
Nox2ds-tat given at reperfusion dose-
dependently attenuated IR induced cardiac
contractile dysfunction and recovered post-
reperfused LVDP to 47 plusmn 7 (5 μM n=7) 69 plusmn
13 (10 μM n=6 plt005) 68 plusmn 7 (40 μM
n=6 plt005) and 77 plusmn 7 (80 μM n=6 plt005)
of initial LVDP at 45 min post-reperfusion when
compared to control IR hearts that only
recovered to 46 plusmn 6 (n=14) (Table 1) Initial
baseline values for cardiac function parameters
did not differ significantly among groups
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 215
Table 1 Cardiac function parameters among different experimental groups
Cardiac Parameters Control IR
(n=14)
IR +
Nox2ds-tat 5
μM
(n=7)
IR +
Nox2ds-tat
10 μM
(n=6)
IR +
Nox2ds-tat
40 μM
(n=6)
IR +
Nox2ds-tat
80 μM
(n=6)
Initial LVDP (mmHg) 87plusmn35 91plusmn31 89plusmn34 92plusmn39 88plusmn33
Final LVDP (mmHg) 40plusmn50 43plusmn66 61plusmn121 63plusmn62 68plusmn62
Initial LVESP (mmHg) 99plusmn38 101plusmn39 105plusmn48 103plusmn46 101plusmn37
Final LVESP (mmHg) 100plusmn53 108plusmn57 107plusmn119 110plusmn75 124plusmn97
Initial LVEDP (mmHg) 11plusmn10 94plusmn13 16plusmn20 11plusmn13 13plusmn07
Final LVEDP(mmHg) 61plusmn51 64plusmn42 46plusmn61 47plusmn75
56plusmn56
Initial +dPdtmax (mmHgsec) 2259plusmn71 2335plusmn61 2333plusmn66 2346plusmn77 2277plusmn79
Final +dPdtmax (mmHgsec) 894plusmn105 832plusmn124 1285plusmn232 1258plusmn120
1413plusmn100
Initial -dPdtmin (mmHgsec) -1510plusmn67 -1616plusmn98 -1607plusmn84 -1550plusmn124 -1524plusmn106
Final -dPdtmin (mmHgsec) -704plusmn65 -660plusmn136 -879plusmn187 -939plusmn108 -1116plusmn146
Initial coronary flow
(mlmin) 162plusmn12 144plusmn064 181plusmn14 180plusmn25 152plusmn10
Final coronary flow (mlmin) 79plusmn064 79plusmn070 92plusmn15 93plusmn17 95plusmn15
Initial Heart Rate (BPM) 271plusmn128 277plusmn123 280plusmn78 272plusmn88 265plusmn32
Final Heart Rate (BPM) 246plusmn188 234plusmn199 230plusmn105 249plusmn136 237plusmn119
plt005 vs control IR final cardiac function parameters plt005
plt001 vs Nox2ds-tat 5μM
Similarly Nox2ds-tat reduced infarct
sizearea-at-risk when compared to the IR
control group (Figure 4 Panel A-B) The cross
section for the IR control group shows the
majority of the infarct in the mid-wall area
whereas the Nox2ds-tat treated IR hearts showed
less infarct area (Figure 4 Panel A) Figure 4
Panel B shows the ratio of this tissue at risk
When compared to the IR control group (46 plusmn
2) there was a significant decrease in total
infarct size in hearts treated with Nox2ds-tat 30 plusmn
4 (5 μM) 15 plusmn 14 (10 μM) 23 plusmn 2 (40
microM) and 19 plusmn 16 (80 microM)(plt001 for all
groups) Moreover the 10 μM and 80 μM doses
showed significantly less infarct size compared to
the 5 μM dose (plt001 and plt005 respectively)
suggesting a dose-dependent effect of Nox2ds-tat
on infarct size (Figure 4 Panel A-B)
Other cardiac function parameters are
illustrated in Table 1 Of interest Nox2ds-tat
dose-dependently recovered post-reperfused final
+dPdtmax by 36 plusmn 53 (5 μM) 55 plusmn10 (10
μM) 54 plusmn 4 (40 microM plt005) 62 plusmn 4 (80 microM
plt005) at 45 min reperfusion compared to
control IR hearts that only recovered to 40 plusmn 5
Sham hearts (n=6) were not subjected to IR and
maintained cardiac function parameters and flow
by gt90 and had lt5 infarct sizearea-at- risk
throughout the experimental time-course (data
not shown)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 216
Figure 4 Panel A Representative images of TTC staining and percentage of left ventricular heart tissue at risk among
different experimental groups Viable tissue stains red infarcted tissue did not retain TTC staining Nox2ds-tat dose
dependently attenuated infarct size Panel B Ratio of left ventricular heart tissue at risk for myocardial dysfunction
out of total tissue weight as determined by TTC staining of representative heart sections When compared to the IR
control group which had an infarct size of 46plusmn2 there was a significant decrease in total infarct size in heart sections
treated with Nox2ds-tat Nox2ds-tat dose-dependently attenuated infarct sizes to 30 plusmn 4 (5 μM n=7) 15 plusmn 14 (10
μM n=6) 23 plusmn 2 (40 microM n=6) and 19 plusmn 16 (80 microM n=6) (plt001 vs control tissue n=14) There was also a
significant decrease in infarct size observed in 10 μM (plt001) and 80 microM (plt005) compared to 5 μM Nox2ds-tat
(plt001 vs control IR plt005 vs Nox2ds-tat 5 microM plt001 vs Nox2ds-tat 5microM)
33 Hindlimb IR in vivo
331 Nox2ds-tat reduced blood H2O2 levels
The blood H2O2 values in the IR hindlimb of
saline controls significantly increased to 17 plusmn 03
microM (15 min post-reperfusion) above sham values
Thereafter a relative difference of 12-14 μM
between the IR and sham limbs was maintained
throughout the remainder of reperfusion (Figure 5
Panel A) By contrast Nox2ds-tat (41 mgkg
~20 μM in blood) treated animals in the IR
hindlimb did not significantly differ from the
sham hindlimb throughout reperfusion (Figure 5
Panel B) The relative difference in blood H2O2
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant reduction in the IR induced effect on
blood H2O2 by 143 plusmn 02 μM at the end of
reperfusion (Figure 5 Panel C)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 217
Figure 5 Panel A Relative difference in measured blood H2O2 between IR and sham femoral veins in saline controls
The saline IR vein showed a significant increase in blood H2O2 by 17 plusmn 03 microM (5 min) relative to the sham femoral
vein This increase above sham femoral vein values was maintained throughout reperfusion stabilizing at 14 plusmn 02 microM
(45 min) (plt005 plt001 vs sham IR femoral vein) Panel B Relative difference in measured blood H2O2
between IR and sham femoral veins in Nox2ds-tat (41 mgkg ~20 microM) treated rats The IR femoral vein treated with
Nox2ds-tat showed a transient increase in H2O2 when compared to the sham femoral vein However this difference
between the two limbs decreased throughout the remainder of the experiment ending at ~0 microM The measured values
in the two groups were not statistically different at any point in the experiment Panel C Relative difference in
measured blood H2O2 betweenNox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline
values) Nox2ds-tat infusion at reperfusion significantly decreased the blood H2O2 concentration when compared to
rats infused with saline At the end of the 45 min reperfusion period there was a significant reduction of 14 microM
between blood H2O2 measured in Nox2ds-tat 20 microM treated rats relative to saline control (plt005 plt001 vs
saline treated femoral veins)
B
A
C
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 218
A
B
C
Figure 6 Panel A Relative difference in measured blood NO between IR and sham femoral veins in saline controls
Overall the IR femoral vein showed a significant decrease in NO concentration stabilizing at a difference of 90 plusmn 21
nM when compared to the sham femoral vein at the end of the 45 min reperfusion period (plt005 plt001 vs sham
IR femoral vein) Panel B Relative difference in measured blood NO between IR and sham femoral veins in Nox2ds-
tat (41 mgkg ~20 microM) treated rats In Nox2ds-tat treated animals the IR femoral vein resulted in 37 plusmn 12 nM higher
blood NO concentration than the sham limb at the end of the 45 min reperfusion period however this difference was
not statistically significant (plt005 plt001 vs sham IR femoral vein) Panel C Relative difference in measured
blood NO in Nox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline values) Nox2ds-tat
given at reperfusion significantly increased the total blood NO concentration when compared to saline controls At the
end of the 45 min reperfusion period there was a significant increase of 127 nM in blood NO in Nox2ds-tat treated
animals relative to saline controls (plt005 plt001 vs saline treated femoral veins)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 219
332 Nox2ds-tat prevented the decrease in
blood NO levels
In saline controls blood NO levels steadily
decreased in the IR limb throughout the
reperfusion period leading to significantly lower
blood NO bioavailability (90 nM below sham) as
early as 20 min into the reperfusion period
(Figure 6 Panel A) By contrast blood NO in the
IR hindlimb of Nox2ds-tat (41 mgkg ~20 μM
in blood) treated animals did not significantly
differ from sham hindlimb values during the
reperfusion period While a linear trend close to
sham values was observed the Nox2ds-tat treated
grouprsquos blood NO levels did rise to 37 nM above
sham values at the end of reperfusion (Figure 6
Panel B) The relative difference in blood NO
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant increase in the IR induced effect on
NO release by 127 nM at the end of reperfusion
(Figure 6 Panel C)
34 L-NAME induced Leukocyte endothelial
interactions
341 Intravital Microscopy
There was no significant difference among
initial baseline values in leukocyte rolling
adherence and transmigration among all study
groups In all parameters the Krebsrsquo buffer group
did not significantly change from baseline L-
NAME (50 microM) significantly increased leukocyte
rolling adherence and transmigration when
compared to Krebsrsquo at T=120 min by 82 plusmn 11
(plt001) 22 plusmn 5 (plt001) and 21 plusmn 4 (plt001)
respectively (Table 2) Nox2ds-tat (5 microM) did not
significantly attenuate L-NAME induced
leukocyte-endothelial interactions except for final
transmigration (plt005) By contrast Nox2ds-tat
(20 microM) significantly decreased the L-NAME
induced effect by 3-5 fold on all parameters
(plt001) (Table 2)
Table 2 The comparision of initial and final (120 min of reperfusion) leukocyte rolling adherence and transmigration
between Krebsrsquo L-NAME and L-NAME + Nox2ds-tat (5 μM 20 μM) groups in rat mesenteric venules
Intravital microscopy Parameters
Krebsrsquo
(n=6)
L-NAME
(n=5)
L-NAME +
Nox2ds-tat 5 μM
(n=4)
L-NAME +
Nox2ds-tat 20 μM
(n=6)
Initial Rolling (numbermin) 8plusmn3 11plusmn3 13plusmn3 8plusmn2
Final Rolling (numbermin) 16plusmn5 82plusmn11
72plusmn13 16plusmn6++
Initial Adherence (number100um) 2plusmn2 1plusmn0 3plusmn0 1plusmn1
Final Adherence (number100um) 4plusmn2 22plusmn5 17plusmn1 8plusmn4
Initial Transmigration (number) 1plusmn0 1plusmn0 1plusmn0 1plusmn0
Final Transmigration (number) 1plusmn0 21plusmn4 9plusmn1
4plusmn2
Plt005 Plt001 compared to Krebsrsquo groupPlt005
Plt001 compared to L-NAME
++Plt001 compared to L-
NAME + Nox2ds-tat 5 μM
342 HampE Staining
The Krebsrsquocontrol group exhibited only 73 plusmn
33 and 62 plusmn 30 leukocytesmm2 of mesenteric
tissue These values represent basal numbers of
leukocyte adherence and transmigration in this
tissue bed respectively L-NAME treatment
exhibited a marked increase (~4-fold) in
leukocyte vascular adherence and transmigration
compared to Krebsrsquo buffer (299 plusmn 23 and 271 plusmn
26 leukocytesmm2 of tissue respectively plt001
for both values) Nox2ds-tat dose-dependently
attenuated the L-NAME induced effect on
leukocyte adherence (199 plusmn 13 for 5 microM at
plt005 and 83 plusmn 9 for 20 microM plt001) and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
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Myocardial reperfusion injury New England
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Myocardial ischemia-reperfusion injury a
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101172JCI62874
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4 Ago T Kuroda J Kamouchi M
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Pathophysiological Roles of NADPH
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101161CIRCRESAHA107162800
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7 Fabian R H Perez-Polo J R amp Kent T
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Michael L H Youker K A Bressler R
B Mendoza L H amp Entman M L
(1998) Resident cardiac mast cells
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9 Nolly M B Caldiz C I Yeves A M
Villa-Abrille M C Morgan P E
Mondaca N A amp Ennis I L (2014)
The signaling pathway for aldosterone-
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superoxide anion in the myocardium
Journal of Molecular and Cellular
Cardiology 67 60-68 DOI
101016jyjmcc201312004
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10 Kleniewska P Piechota A Skibska B amp
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60(4) 277-294 DOI 101007s00005-012-
0176-z
11 Laurindo F R Araujo T L amp Abrahao T
B (2014) Nox NADPH oxidases and the
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12 Li J M amp Shah A M (2003) Mechanism
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Activation by Angiotensin II ROLE OF THE
p47 phox SUBUNIT Journal of Biological
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101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
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14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 226
phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
Biochemistry 46(51) 14771-14781 DOI
101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
Immunology 33(5) 1328-1333 DOI
101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
inhibition attenuates myocardial infarction
induced heart failure and is associated with a
reduction of fibrosis and pro‐inflammatory
responses Journal of Cellular and
Molecular Medicine 15(8) 1769-1777 DOI
101111j1582-4934201001174x
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 215
Table 1 Cardiac function parameters among different experimental groups
Cardiac Parameters Control IR
(n=14)
IR +
Nox2ds-tat 5
μM
(n=7)
IR +
Nox2ds-tat
10 μM
(n=6)
IR +
Nox2ds-tat
40 μM
(n=6)
IR +
Nox2ds-tat
80 μM
(n=6)
Initial LVDP (mmHg) 87plusmn35 91plusmn31 89plusmn34 92plusmn39 88plusmn33
Final LVDP (mmHg) 40plusmn50 43plusmn66 61plusmn121 63plusmn62 68plusmn62
Initial LVESP (mmHg) 99plusmn38 101plusmn39 105plusmn48 103plusmn46 101plusmn37
Final LVESP (mmHg) 100plusmn53 108plusmn57 107plusmn119 110plusmn75 124plusmn97
Initial LVEDP (mmHg) 11plusmn10 94plusmn13 16plusmn20 11plusmn13 13plusmn07
Final LVEDP(mmHg) 61plusmn51 64plusmn42 46plusmn61 47plusmn75
56plusmn56
Initial +dPdtmax (mmHgsec) 2259plusmn71 2335plusmn61 2333plusmn66 2346plusmn77 2277plusmn79
Final +dPdtmax (mmHgsec) 894plusmn105 832plusmn124 1285plusmn232 1258plusmn120
1413plusmn100
Initial -dPdtmin (mmHgsec) -1510plusmn67 -1616plusmn98 -1607plusmn84 -1550plusmn124 -1524plusmn106
Final -dPdtmin (mmHgsec) -704plusmn65 -660plusmn136 -879plusmn187 -939plusmn108 -1116plusmn146
Initial coronary flow
(mlmin) 162plusmn12 144plusmn064 181plusmn14 180plusmn25 152plusmn10
Final coronary flow (mlmin) 79plusmn064 79plusmn070 92plusmn15 93plusmn17 95plusmn15
Initial Heart Rate (BPM) 271plusmn128 277plusmn123 280plusmn78 272plusmn88 265plusmn32
Final Heart Rate (BPM) 246plusmn188 234plusmn199 230plusmn105 249plusmn136 237plusmn119
plt005 vs control IR final cardiac function parameters plt005
plt001 vs Nox2ds-tat 5μM
Similarly Nox2ds-tat reduced infarct
sizearea-at-risk when compared to the IR
control group (Figure 4 Panel A-B) The cross
section for the IR control group shows the
majority of the infarct in the mid-wall area
whereas the Nox2ds-tat treated IR hearts showed
less infarct area (Figure 4 Panel A) Figure 4
Panel B shows the ratio of this tissue at risk
When compared to the IR control group (46 plusmn
2) there was a significant decrease in total
infarct size in hearts treated with Nox2ds-tat 30 plusmn
4 (5 μM) 15 plusmn 14 (10 μM) 23 plusmn 2 (40
microM) and 19 plusmn 16 (80 microM)(plt001 for all
groups) Moreover the 10 μM and 80 μM doses
showed significantly less infarct size compared to
the 5 μM dose (plt001 and plt005 respectively)
suggesting a dose-dependent effect of Nox2ds-tat
on infarct size (Figure 4 Panel A-B)
Other cardiac function parameters are
illustrated in Table 1 Of interest Nox2ds-tat
dose-dependently recovered post-reperfused final
+dPdtmax by 36 plusmn 53 (5 μM) 55 plusmn10 (10
μM) 54 plusmn 4 (40 microM plt005) 62 plusmn 4 (80 microM
plt005) at 45 min reperfusion compared to
control IR hearts that only recovered to 40 plusmn 5
Sham hearts (n=6) were not subjected to IR and
maintained cardiac function parameters and flow
by gt90 and had lt5 infarct sizearea-at- risk
throughout the experimental time-course (data
not shown)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 216
Figure 4 Panel A Representative images of TTC staining and percentage of left ventricular heart tissue at risk among
different experimental groups Viable tissue stains red infarcted tissue did not retain TTC staining Nox2ds-tat dose
dependently attenuated infarct size Panel B Ratio of left ventricular heart tissue at risk for myocardial dysfunction
out of total tissue weight as determined by TTC staining of representative heart sections When compared to the IR
control group which had an infarct size of 46plusmn2 there was a significant decrease in total infarct size in heart sections
treated with Nox2ds-tat Nox2ds-tat dose-dependently attenuated infarct sizes to 30 plusmn 4 (5 μM n=7) 15 plusmn 14 (10
μM n=6) 23 plusmn 2 (40 microM n=6) and 19 plusmn 16 (80 microM n=6) (plt001 vs control tissue n=14) There was also a
significant decrease in infarct size observed in 10 μM (plt001) and 80 microM (plt005) compared to 5 μM Nox2ds-tat
(plt001 vs control IR plt005 vs Nox2ds-tat 5 microM plt001 vs Nox2ds-tat 5microM)
33 Hindlimb IR in vivo
331 Nox2ds-tat reduced blood H2O2 levels
The blood H2O2 values in the IR hindlimb of
saline controls significantly increased to 17 plusmn 03
microM (15 min post-reperfusion) above sham values
Thereafter a relative difference of 12-14 μM
between the IR and sham limbs was maintained
throughout the remainder of reperfusion (Figure 5
Panel A) By contrast Nox2ds-tat (41 mgkg
~20 μM in blood) treated animals in the IR
hindlimb did not significantly differ from the
sham hindlimb throughout reperfusion (Figure 5
Panel B) The relative difference in blood H2O2
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant reduction in the IR induced effect on
blood H2O2 by 143 plusmn 02 μM at the end of
reperfusion (Figure 5 Panel C)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 217
Figure 5 Panel A Relative difference in measured blood H2O2 between IR and sham femoral veins in saline controls
The saline IR vein showed a significant increase in blood H2O2 by 17 plusmn 03 microM (5 min) relative to the sham femoral
vein This increase above sham femoral vein values was maintained throughout reperfusion stabilizing at 14 plusmn 02 microM
(45 min) (plt005 plt001 vs sham IR femoral vein) Panel B Relative difference in measured blood H2O2
between IR and sham femoral veins in Nox2ds-tat (41 mgkg ~20 microM) treated rats The IR femoral vein treated with
Nox2ds-tat showed a transient increase in H2O2 when compared to the sham femoral vein However this difference
between the two limbs decreased throughout the remainder of the experiment ending at ~0 microM The measured values
in the two groups were not statistically different at any point in the experiment Panel C Relative difference in
measured blood H2O2 betweenNox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline
values) Nox2ds-tat infusion at reperfusion significantly decreased the blood H2O2 concentration when compared to
rats infused with saline At the end of the 45 min reperfusion period there was a significant reduction of 14 microM
between blood H2O2 measured in Nox2ds-tat 20 microM treated rats relative to saline control (plt005 plt001 vs
saline treated femoral veins)
B
A
C
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 218
A
B
C
Figure 6 Panel A Relative difference in measured blood NO between IR and sham femoral veins in saline controls
Overall the IR femoral vein showed a significant decrease in NO concentration stabilizing at a difference of 90 plusmn 21
nM when compared to the sham femoral vein at the end of the 45 min reperfusion period (plt005 plt001 vs sham
IR femoral vein) Panel B Relative difference in measured blood NO between IR and sham femoral veins in Nox2ds-
tat (41 mgkg ~20 microM) treated rats In Nox2ds-tat treated animals the IR femoral vein resulted in 37 plusmn 12 nM higher
blood NO concentration than the sham limb at the end of the 45 min reperfusion period however this difference was
not statistically significant (plt005 plt001 vs sham IR femoral vein) Panel C Relative difference in measured
blood NO in Nox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline values) Nox2ds-tat
given at reperfusion significantly increased the total blood NO concentration when compared to saline controls At the
end of the 45 min reperfusion period there was a significant increase of 127 nM in blood NO in Nox2ds-tat treated
animals relative to saline controls (plt005 plt001 vs saline treated femoral veins)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 219
332 Nox2ds-tat prevented the decrease in
blood NO levels
In saline controls blood NO levels steadily
decreased in the IR limb throughout the
reperfusion period leading to significantly lower
blood NO bioavailability (90 nM below sham) as
early as 20 min into the reperfusion period
(Figure 6 Panel A) By contrast blood NO in the
IR hindlimb of Nox2ds-tat (41 mgkg ~20 μM
in blood) treated animals did not significantly
differ from sham hindlimb values during the
reperfusion period While a linear trend close to
sham values was observed the Nox2ds-tat treated
grouprsquos blood NO levels did rise to 37 nM above
sham values at the end of reperfusion (Figure 6
Panel B) The relative difference in blood NO
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant increase in the IR induced effect on
NO release by 127 nM at the end of reperfusion
(Figure 6 Panel C)
34 L-NAME induced Leukocyte endothelial
interactions
341 Intravital Microscopy
There was no significant difference among
initial baseline values in leukocyte rolling
adherence and transmigration among all study
groups In all parameters the Krebsrsquo buffer group
did not significantly change from baseline L-
NAME (50 microM) significantly increased leukocyte
rolling adherence and transmigration when
compared to Krebsrsquo at T=120 min by 82 plusmn 11
(plt001) 22 plusmn 5 (plt001) and 21 plusmn 4 (plt001)
respectively (Table 2) Nox2ds-tat (5 microM) did not
significantly attenuate L-NAME induced
leukocyte-endothelial interactions except for final
transmigration (plt005) By contrast Nox2ds-tat
(20 microM) significantly decreased the L-NAME
induced effect by 3-5 fold on all parameters
(plt001) (Table 2)
Table 2 The comparision of initial and final (120 min of reperfusion) leukocyte rolling adherence and transmigration
between Krebsrsquo L-NAME and L-NAME + Nox2ds-tat (5 μM 20 μM) groups in rat mesenteric venules
Intravital microscopy Parameters
Krebsrsquo
(n=6)
L-NAME
(n=5)
L-NAME +
Nox2ds-tat 5 μM
(n=4)
L-NAME +
Nox2ds-tat 20 μM
(n=6)
Initial Rolling (numbermin) 8plusmn3 11plusmn3 13plusmn3 8plusmn2
Final Rolling (numbermin) 16plusmn5 82plusmn11
72plusmn13 16plusmn6++
Initial Adherence (number100um) 2plusmn2 1plusmn0 3plusmn0 1plusmn1
Final Adherence (number100um) 4plusmn2 22plusmn5 17plusmn1 8plusmn4
Initial Transmigration (number) 1plusmn0 1plusmn0 1plusmn0 1plusmn0
Final Transmigration (number) 1plusmn0 21plusmn4 9plusmn1
4plusmn2
Plt005 Plt001 compared to Krebsrsquo groupPlt005
Plt001 compared to L-NAME
++Plt001 compared to L-
NAME + Nox2ds-tat 5 μM
342 HampE Staining
The Krebsrsquocontrol group exhibited only 73 plusmn
33 and 62 plusmn 30 leukocytesmm2 of mesenteric
tissue These values represent basal numbers of
leukocyte adherence and transmigration in this
tissue bed respectively L-NAME treatment
exhibited a marked increase (~4-fold) in
leukocyte vascular adherence and transmigration
compared to Krebsrsquo buffer (299 plusmn 23 and 271 plusmn
26 leukocytesmm2 of tissue respectively plt001
for both values) Nox2ds-tat dose-dependently
attenuated the L-NAME induced effect on
leukocyte adherence (199 plusmn 13 for 5 microM at
plt005 and 83 plusmn 9 for 20 microM plt001) and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
References
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Myocardial reperfusion injury New England
Journal of Medicine 357(11) 1121-1135
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Myocardial ischemia-reperfusion injury a
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101172JCI62874
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Sadoshima J amp Kitazono T (2011)
Pathophysiological Roles of NADPH
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101161CIRCRESAHA107162800
6 Duda M Konior A Klemenska E amp
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101016jyjmcc200610014
7 Fabian R H Perez-Polo J R amp Kent T
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101152ajpheart003012007
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Michael L H Youker K A Bressler R
B Mendoza L H amp Entman M L
(1998) Resident cardiac mast cells
degranulate and release preformed TNF-α
initiating the cytokine cascade in
experimental canine myocardial
ischemiareperfusion Circulation 98(7)
699-710 DOI 10116101CIR987699
9 Nolly M B Caldiz C I Yeves A M
Villa-Abrille M C Morgan P E
Mondaca N A amp Ennis I L (2014)
The signaling pathway for aldosterone-
induced mitochondrial production of
superoxide anion in the myocardium
Journal of Molecular and Cellular
Cardiology 67 60-68 DOI
101016jyjmcc201312004
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10 Kleniewska P Piechota A Skibska B amp
Gorąca A (2012) The NADPH oxidase
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immunologiae et therapiae experimentalis
60(4) 277-294 DOI 101007s00005-012-
0176-z
11 Laurindo F R Araujo T L amp Abrahao T
B (2014) Nox NADPH oxidases and the
endoplasmic reticulum Antioxidants amp
Redox Signaling 20(17) 2755-2775 DOI
101089ars20135605
12 Li J M amp Shah A M (2003) Mechanism
of Endothelial Cell NADPH Oxidase
Activation by Angiotensin II ROLE OF THE
p47 phox SUBUNIT Journal of Biological
Chemistry 278(14) 12094-12100 DOI
101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
attenuated neurohumoral excitation in heart
failure a role for superoxide and nitric oxide
Basic Research in Cardiology 106(2) 273-
286 DOI 101007s00395-010-0146-8
14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
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phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
Biochemistry 46(51) 14771-14781 DOI
101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
Immunology 33(5) 1328-1333 DOI
101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
inhibition attenuates myocardial infarction
induced heart failure and is associated with a
reduction of fibrosis and pro‐inflammatory
responses Journal of Cellular and
Molecular Medicine 15(8) 1769-1777 DOI
101111j1582-4934201001174x
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 216
Figure 4 Panel A Representative images of TTC staining and percentage of left ventricular heart tissue at risk among
different experimental groups Viable tissue stains red infarcted tissue did not retain TTC staining Nox2ds-tat dose
dependently attenuated infarct size Panel B Ratio of left ventricular heart tissue at risk for myocardial dysfunction
out of total tissue weight as determined by TTC staining of representative heart sections When compared to the IR
control group which had an infarct size of 46plusmn2 there was a significant decrease in total infarct size in heart sections
treated with Nox2ds-tat Nox2ds-tat dose-dependently attenuated infarct sizes to 30 plusmn 4 (5 μM n=7) 15 plusmn 14 (10
μM n=6) 23 plusmn 2 (40 microM n=6) and 19 plusmn 16 (80 microM n=6) (plt001 vs control tissue n=14) There was also a
significant decrease in infarct size observed in 10 μM (plt001) and 80 microM (plt005) compared to 5 μM Nox2ds-tat
(plt001 vs control IR plt005 vs Nox2ds-tat 5 microM plt001 vs Nox2ds-tat 5microM)
33 Hindlimb IR in vivo
331 Nox2ds-tat reduced blood H2O2 levels
The blood H2O2 values in the IR hindlimb of
saline controls significantly increased to 17 plusmn 03
microM (15 min post-reperfusion) above sham values
Thereafter a relative difference of 12-14 μM
between the IR and sham limbs was maintained
throughout the remainder of reperfusion (Figure 5
Panel A) By contrast Nox2ds-tat (41 mgkg
~20 μM in blood) treated animals in the IR
hindlimb did not significantly differ from the
sham hindlimb throughout reperfusion (Figure 5
Panel B) The relative difference in blood H2O2
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant reduction in the IR induced effect on
blood H2O2 by 143 plusmn 02 μM at the end of
reperfusion (Figure 5 Panel C)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 217
Figure 5 Panel A Relative difference in measured blood H2O2 between IR and sham femoral veins in saline controls
The saline IR vein showed a significant increase in blood H2O2 by 17 plusmn 03 microM (5 min) relative to the sham femoral
vein This increase above sham femoral vein values was maintained throughout reperfusion stabilizing at 14 plusmn 02 microM
(45 min) (plt005 plt001 vs sham IR femoral vein) Panel B Relative difference in measured blood H2O2
between IR and sham femoral veins in Nox2ds-tat (41 mgkg ~20 microM) treated rats The IR femoral vein treated with
Nox2ds-tat showed a transient increase in H2O2 when compared to the sham femoral vein However this difference
between the two limbs decreased throughout the remainder of the experiment ending at ~0 microM The measured values
in the two groups were not statistically different at any point in the experiment Panel C Relative difference in
measured blood H2O2 betweenNox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline
values) Nox2ds-tat infusion at reperfusion significantly decreased the blood H2O2 concentration when compared to
rats infused with saline At the end of the 45 min reperfusion period there was a significant reduction of 14 microM
between blood H2O2 measured in Nox2ds-tat 20 microM treated rats relative to saline control (plt005 plt001 vs
saline treated femoral veins)
B
A
C
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 218
A
B
C
Figure 6 Panel A Relative difference in measured blood NO between IR and sham femoral veins in saline controls
Overall the IR femoral vein showed a significant decrease in NO concentration stabilizing at a difference of 90 plusmn 21
nM when compared to the sham femoral vein at the end of the 45 min reperfusion period (plt005 plt001 vs sham
IR femoral vein) Panel B Relative difference in measured blood NO between IR and sham femoral veins in Nox2ds-
tat (41 mgkg ~20 microM) treated rats In Nox2ds-tat treated animals the IR femoral vein resulted in 37 plusmn 12 nM higher
blood NO concentration than the sham limb at the end of the 45 min reperfusion period however this difference was
not statistically significant (plt005 plt001 vs sham IR femoral vein) Panel C Relative difference in measured
blood NO in Nox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline values) Nox2ds-tat
given at reperfusion significantly increased the total blood NO concentration when compared to saline controls At the
end of the 45 min reperfusion period there was a significant increase of 127 nM in blood NO in Nox2ds-tat treated
animals relative to saline controls (plt005 plt001 vs saline treated femoral veins)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 219
332 Nox2ds-tat prevented the decrease in
blood NO levels
In saline controls blood NO levels steadily
decreased in the IR limb throughout the
reperfusion period leading to significantly lower
blood NO bioavailability (90 nM below sham) as
early as 20 min into the reperfusion period
(Figure 6 Panel A) By contrast blood NO in the
IR hindlimb of Nox2ds-tat (41 mgkg ~20 μM
in blood) treated animals did not significantly
differ from sham hindlimb values during the
reperfusion period While a linear trend close to
sham values was observed the Nox2ds-tat treated
grouprsquos blood NO levels did rise to 37 nM above
sham values at the end of reperfusion (Figure 6
Panel B) The relative difference in blood NO
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant increase in the IR induced effect on
NO release by 127 nM at the end of reperfusion
(Figure 6 Panel C)
34 L-NAME induced Leukocyte endothelial
interactions
341 Intravital Microscopy
There was no significant difference among
initial baseline values in leukocyte rolling
adherence and transmigration among all study
groups In all parameters the Krebsrsquo buffer group
did not significantly change from baseline L-
NAME (50 microM) significantly increased leukocyte
rolling adherence and transmigration when
compared to Krebsrsquo at T=120 min by 82 plusmn 11
(plt001) 22 plusmn 5 (plt001) and 21 plusmn 4 (plt001)
respectively (Table 2) Nox2ds-tat (5 microM) did not
significantly attenuate L-NAME induced
leukocyte-endothelial interactions except for final
transmigration (plt005) By contrast Nox2ds-tat
(20 microM) significantly decreased the L-NAME
induced effect by 3-5 fold on all parameters
(plt001) (Table 2)
Table 2 The comparision of initial and final (120 min of reperfusion) leukocyte rolling adherence and transmigration
between Krebsrsquo L-NAME and L-NAME + Nox2ds-tat (5 μM 20 μM) groups in rat mesenteric venules
Intravital microscopy Parameters
Krebsrsquo
(n=6)
L-NAME
(n=5)
L-NAME +
Nox2ds-tat 5 μM
(n=4)
L-NAME +
Nox2ds-tat 20 μM
(n=6)
Initial Rolling (numbermin) 8plusmn3 11plusmn3 13plusmn3 8plusmn2
Final Rolling (numbermin) 16plusmn5 82plusmn11
72plusmn13 16plusmn6++
Initial Adherence (number100um) 2plusmn2 1plusmn0 3plusmn0 1plusmn1
Final Adherence (number100um) 4plusmn2 22plusmn5 17plusmn1 8plusmn4
Initial Transmigration (number) 1plusmn0 1plusmn0 1plusmn0 1plusmn0
Final Transmigration (number) 1plusmn0 21plusmn4 9plusmn1
4plusmn2
Plt005 Plt001 compared to Krebsrsquo groupPlt005
Plt001 compared to L-NAME
++Plt001 compared to L-
NAME + Nox2ds-tat 5 μM
342 HampE Staining
The Krebsrsquocontrol group exhibited only 73 plusmn
33 and 62 plusmn 30 leukocytesmm2 of mesenteric
tissue These values represent basal numbers of
leukocyte adherence and transmigration in this
tissue bed respectively L-NAME treatment
exhibited a marked increase (~4-fold) in
leukocyte vascular adherence and transmigration
compared to Krebsrsquo buffer (299 plusmn 23 and 271 plusmn
26 leukocytesmm2 of tissue respectively plt001
for both values) Nox2ds-tat dose-dependently
attenuated the L-NAME induced effect on
leukocyte adherence (199 plusmn 13 for 5 microM at
plt005 and 83 plusmn 9 for 20 microM plt001) and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
References
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Myocardial reperfusion injury New England
Journal of Medicine 357(11) 1121-1135
DOI 101056NEJMra071667
2 Hausenloy D J amp Yellon D M (2013)
Myocardial ischemia-reperfusion injury a
neglected therapeutic target Journal of
Clinical Investigation 123(1) 92-100 DOI
101172JCI62874
3 Lefer A M amp Lefer D J (1996) The role
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reperfusion Cardiovascular Research 32(4)
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6363(96)00073-9
4 Ago T Kuroda J Kamouchi M
Sadoshima J amp Kitazono T (2011)
Pathophysiological Roles of NADPH
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101253circjCJ-11-0388
5 Doughan A K Harrison D G amp Dikalov
S I (2008) Molecular Mechanisms of
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Dysfunction Linking Mitochondrial
Oxidative Damage and Vascular Endothelial
Dysfunction Circulation research 102(4)
488-496 DOI
101161CIRCRESAHA107162800
6 Duda M Konior A Klemenska E amp
Beręsewicz A (2007) Preconditioning
protects endothelium by preventing ET-1-
induced activation of NADPH oxidase and
xanthine oxidase in post-ischemic heart
Journal of Molecular and Cellular
Cardiology 42(2) 400-410 DOI
101016jyjmcc200610014
7 Fabian R H Perez-Polo J R amp Kent T
A (2008) Perivascular nitric oxide and
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ischemia American Journal of Physiology-
Heart and Circulatory Physiology 295(4)
H1809-H1814 DOI
101152ajpheart003012007
8 Frangogiannis N G Lindsey M L
Michael L H Youker K A Bressler R
B Mendoza L H amp Entman M L
(1998) Resident cardiac mast cells
degranulate and release preformed TNF-α
initiating the cytokine cascade in
experimental canine myocardial
ischemiareperfusion Circulation 98(7)
699-710 DOI 10116101CIR987699
9 Nolly M B Caldiz C I Yeves A M
Villa-Abrille M C Morgan P E
Mondaca N A amp Ennis I L (2014)
The signaling pathway for aldosterone-
induced mitochondrial production of
superoxide anion in the myocardium
Journal of Molecular and Cellular
Cardiology 67 60-68 DOI
101016jyjmcc201312004
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 225
10 Kleniewska P Piechota A Skibska B amp
Gorąca A (2012) The NADPH oxidase
family and its inhibitors Archivum
immunologiae et therapiae experimentalis
60(4) 277-294 DOI 101007s00005-012-
0176-z
11 Laurindo F R Araujo T L amp Abrahao T
B (2014) Nox NADPH oxidases and the
endoplasmic reticulum Antioxidants amp
Redox Signaling 20(17) 2755-2775 DOI
101089ars20135605
12 Li J M amp Shah A M (2003) Mechanism
of Endothelial Cell NADPH Oxidase
Activation by Angiotensin II ROLE OF THE
p47 phox SUBUNIT Journal of Biological
Chemistry 278(14) 12094-12100 DOI
101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
attenuated neurohumoral excitation in heart
failure a role for superoxide and nitric oxide
Basic Research in Cardiology 106(2) 273-
286 DOI 101007s00395-010-0146-8
14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 226
phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
Biochemistry 46(51) 14771-14781 DOI
101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
Immunology 33(5) 1328-1333 DOI
101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
inhibition attenuates myocardial infarction
induced heart failure and is associated with a
reduction of fibrosis and pro‐inflammatory
responses Journal of Cellular and
Molecular Medicine 15(8) 1769-1777 DOI
101111j1582-4934201001174x
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 217
Figure 5 Panel A Relative difference in measured blood H2O2 between IR and sham femoral veins in saline controls
The saline IR vein showed a significant increase in blood H2O2 by 17 plusmn 03 microM (5 min) relative to the sham femoral
vein This increase above sham femoral vein values was maintained throughout reperfusion stabilizing at 14 plusmn 02 microM
(45 min) (plt005 plt001 vs sham IR femoral vein) Panel B Relative difference in measured blood H2O2
between IR and sham femoral veins in Nox2ds-tat (41 mgkg ~20 microM) treated rats The IR femoral vein treated with
Nox2ds-tat showed a transient increase in H2O2 when compared to the sham femoral vein However this difference
between the two limbs decreased throughout the remainder of the experiment ending at ~0 microM The measured values
in the two groups were not statistically different at any point in the experiment Panel C Relative difference in
measured blood H2O2 betweenNox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline
values) Nox2ds-tat infusion at reperfusion significantly decreased the blood H2O2 concentration when compared to
rats infused with saline At the end of the 45 min reperfusion period there was a significant reduction of 14 microM
between blood H2O2 measured in Nox2ds-tat 20 microM treated rats relative to saline control (plt005 plt001 vs
saline treated femoral veins)
B
A
C
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 218
A
B
C
Figure 6 Panel A Relative difference in measured blood NO between IR and sham femoral veins in saline controls
Overall the IR femoral vein showed a significant decrease in NO concentration stabilizing at a difference of 90 plusmn 21
nM when compared to the sham femoral vein at the end of the 45 min reperfusion period (plt005 plt001 vs sham
IR femoral vein) Panel B Relative difference in measured blood NO between IR and sham femoral veins in Nox2ds-
tat (41 mgkg ~20 microM) treated rats In Nox2ds-tat treated animals the IR femoral vein resulted in 37 plusmn 12 nM higher
blood NO concentration than the sham limb at the end of the 45 min reperfusion period however this difference was
not statistically significant (plt005 plt001 vs sham IR femoral vein) Panel C Relative difference in measured
blood NO in Nox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline values) Nox2ds-tat
given at reperfusion significantly increased the total blood NO concentration when compared to saline controls At the
end of the 45 min reperfusion period there was a significant increase of 127 nM in blood NO in Nox2ds-tat treated
animals relative to saline controls (plt005 plt001 vs saline treated femoral veins)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 219
332 Nox2ds-tat prevented the decrease in
blood NO levels
In saline controls blood NO levels steadily
decreased in the IR limb throughout the
reperfusion period leading to significantly lower
blood NO bioavailability (90 nM below sham) as
early as 20 min into the reperfusion period
(Figure 6 Panel A) By contrast blood NO in the
IR hindlimb of Nox2ds-tat (41 mgkg ~20 μM
in blood) treated animals did not significantly
differ from sham hindlimb values during the
reperfusion period While a linear trend close to
sham values was observed the Nox2ds-tat treated
grouprsquos blood NO levels did rise to 37 nM above
sham values at the end of reperfusion (Figure 6
Panel B) The relative difference in blood NO
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant increase in the IR induced effect on
NO release by 127 nM at the end of reperfusion
(Figure 6 Panel C)
34 L-NAME induced Leukocyte endothelial
interactions
341 Intravital Microscopy
There was no significant difference among
initial baseline values in leukocyte rolling
adherence and transmigration among all study
groups In all parameters the Krebsrsquo buffer group
did not significantly change from baseline L-
NAME (50 microM) significantly increased leukocyte
rolling adherence and transmigration when
compared to Krebsrsquo at T=120 min by 82 plusmn 11
(plt001) 22 plusmn 5 (plt001) and 21 plusmn 4 (plt001)
respectively (Table 2) Nox2ds-tat (5 microM) did not
significantly attenuate L-NAME induced
leukocyte-endothelial interactions except for final
transmigration (plt005) By contrast Nox2ds-tat
(20 microM) significantly decreased the L-NAME
induced effect by 3-5 fold on all parameters
(plt001) (Table 2)
Table 2 The comparision of initial and final (120 min of reperfusion) leukocyte rolling adherence and transmigration
between Krebsrsquo L-NAME and L-NAME + Nox2ds-tat (5 μM 20 μM) groups in rat mesenteric venules
Intravital microscopy Parameters
Krebsrsquo
(n=6)
L-NAME
(n=5)
L-NAME +
Nox2ds-tat 5 μM
(n=4)
L-NAME +
Nox2ds-tat 20 μM
(n=6)
Initial Rolling (numbermin) 8plusmn3 11plusmn3 13plusmn3 8plusmn2
Final Rolling (numbermin) 16plusmn5 82plusmn11
72plusmn13 16plusmn6++
Initial Adherence (number100um) 2plusmn2 1plusmn0 3plusmn0 1plusmn1
Final Adherence (number100um) 4plusmn2 22plusmn5 17plusmn1 8plusmn4
Initial Transmigration (number) 1plusmn0 1plusmn0 1plusmn0 1plusmn0
Final Transmigration (number) 1plusmn0 21plusmn4 9plusmn1
4plusmn2
Plt005 Plt001 compared to Krebsrsquo groupPlt005
Plt001 compared to L-NAME
++Plt001 compared to L-
NAME + Nox2ds-tat 5 μM
342 HampE Staining
The Krebsrsquocontrol group exhibited only 73 plusmn
33 and 62 plusmn 30 leukocytesmm2 of mesenteric
tissue These values represent basal numbers of
leukocyte adherence and transmigration in this
tissue bed respectively L-NAME treatment
exhibited a marked increase (~4-fold) in
leukocyte vascular adherence and transmigration
compared to Krebsrsquo buffer (299 plusmn 23 and 271 plusmn
26 leukocytesmm2 of tissue respectively plt001
for both values) Nox2ds-tat dose-dependently
attenuated the L-NAME induced effect on
leukocyte adherence (199 plusmn 13 for 5 microM at
plt005 and 83 plusmn 9 for 20 microM plt001) and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
References
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Myocardial reperfusion injury New England
Journal of Medicine 357(11) 1121-1135
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2 Hausenloy D J amp Yellon D M (2013)
Myocardial ischemia-reperfusion injury a
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Clinical Investigation 123(1) 92-100 DOI
101172JCI62874
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Sadoshima J amp Kitazono T (2011)
Pathophysiological Roles of NADPH
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5 Doughan A K Harrison D G amp Dikalov
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101161CIRCRESAHA107162800
6 Duda M Konior A Klemenska E amp
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101016jyjmcc200610014
7 Fabian R H Perez-Polo J R amp Kent T
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101152ajpheart003012007
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Michael L H Youker K A Bressler R
B Mendoza L H amp Entman M L
(1998) Resident cardiac mast cells
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Villa-Abrille M C Morgan P E
Mondaca N A amp Ennis I L (2014)
The signaling pathway for aldosterone-
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Journal of Molecular and Cellular
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101016jyjmcc201312004
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10 Kleniewska P Piechota A Skibska B amp
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60(4) 277-294 DOI 101007s00005-012-
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11 Laurindo F R Araujo T L amp Abrahao T
B (2014) Nox NADPH oxidases and the
endoplasmic reticulum Antioxidants amp
Redox Signaling 20(17) 2755-2775 DOI
101089ars20135605
12 Li J M amp Shah A M (2003) Mechanism
of Endothelial Cell NADPH Oxidase
Activation by Angiotensin II ROLE OF THE
p47 phox SUBUNIT Journal of Biological
Chemistry 278(14) 12094-12100 DOI
101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
attenuated neurohumoral excitation in heart
failure a role for superoxide and nitric oxide
Basic Research in Cardiology 106(2) 273-
286 DOI 101007s00395-010-0146-8
14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
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phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
Biochemistry 46(51) 14771-14781 DOI
101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
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101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
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101111j1582-4934201001174x
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38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
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39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
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American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
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Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 218
A
B
C
Figure 6 Panel A Relative difference in measured blood NO between IR and sham femoral veins in saline controls
Overall the IR femoral vein showed a significant decrease in NO concentration stabilizing at a difference of 90 plusmn 21
nM when compared to the sham femoral vein at the end of the 45 min reperfusion period (plt005 plt001 vs sham
IR femoral vein) Panel B Relative difference in measured blood NO between IR and sham femoral veins in Nox2ds-
tat (41 mgkg ~20 microM) treated rats In Nox2ds-tat treated animals the IR femoral vein resulted in 37 plusmn 12 nM higher
blood NO concentration than the sham limb at the end of the 45 min reperfusion period however this difference was
not statistically significant (plt005 plt001 vs sham IR femoral vein) Panel C Relative difference in measured
blood NO in Nox2ds-tat (IRNox2ds-tat ndash shamNox2ds-tat values) and saline controls (IRsaline ndash shamsaline values) Nox2ds-tat
given at reperfusion significantly increased the total blood NO concentration when compared to saline controls At the
end of the 45 min reperfusion period there was a significant increase of 127 nM in blood NO in Nox2ds-tat treated
animals relative to saline controls (plt005 plt001 vs saline treated femoral veins)
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 219
332 Nox2ds-tat prevented the decrease in
blood NO levels
In saline controls blood NO levels steadily
decreased in the IR limb throughout the
reperfusion period leading to significantly lower
blood NO bioavailability (90 nM below sham) as
early as 20 min into the reperfusion period
(Figure 6 Panel A) By contrast blood NO in the
IR hindlimb of Nox2ds-tat (41 mgkg ~20 μM
in blood) treated animals did not significantly
differ from sham hindlimb values during the
reperfusion period While a linear trend close to
sham values was observed the Nox2ds-tat treated
grouprsquos blood NO levels did rise to 37 nM above
sham values at the end of reperfusion (Figure 6
Panel B) The relative difference in blood NO
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant increase in the IR induced effect on
NO release by 127 nM at the end of reperfusion
(Figure 6 Panel C)
34 L-NAME induced Leukocyte endothelial
interactions
341 Intravital Microscopy
There was no significant difference among
initial baseline values in leukocyte rolling
adherence and transmigration among all study
groups In all parameters the Krebsrsquo buffer group
did not significantly change from baseline L-
NAME (50 microM) significantly increased leukocyte
rolling adherence and transmigration when
compared to Krebsrsquo at T=120 min by 82 plusmn 11
(plt001) 22 plusmn 5 (plt001) and 21 plusmn 4 (plt001)
respectively (Table 2) Nox2ds-tat (5 microM) did not
significantly attenuate L-NAME induced
leukocyte-endothelial interactions except for final
transmigration (plt005) By contrast Nox2ds-tat
(20 microM) significantly decreased the L-NAME
induced effect by 3-5 fold on all parameters
(plt001) (Table 2)
Table 2 The comparision of initial and final (120 min of reperfusion) leukocyte rolling adherence and transmigration
between Krebsrsquo L-NAME and L-NAME + Nox2ds-tat (5 μM 20 μM) groups in rat mesenteric venules
Intravital microscopy Parameters
Krebsrsquo
(n=6)
L-NAME
(n=5)
L-NAME +
Nox2ds-tat 5 μM
(n=4)
L-NAME +
Nox2ds-tat 20 μM
(n=6)
Initial Rolling (numbermin) 8plusmn3 11plusmn3 13plusmn3 8plusmn2
Final Rolling (numbermin) 16plusmn5 82plusmn11
72plusmn13 16plusmn6++
Initial Adherence (number100um) 2plusmn2 1plusmn0 3plusmn0 1plusmn1
Final Adherence (number100um) 4plusmn2 22plusmn5 17plusmn1 8plusmn4
Initial Transmigration (number) 1plusmn0 1plusmn0 1plusmn0 1plusmn0
Final Transmigration (number) 1plusmn0 21plusmn4 9plusmn1
4plusmn2
Plt005 Plt001 compared to Krebsrsquo groupPlt005
Plt001 compared to L-NAME
++Plt001 compared to L-
NAME + Nox2ds-tat 5 μM
342 HampE Staining
The Krebsrsquocontrol group exhibited only 73 plusmn
33 and 62 plusmn 30 leukocytesmm2 of mesenteric
tissue These values represent basal numbers of
leukocyte adherence and transmigration in this
tissue bed respectively L-NAME treatment
exhibited a marked increase (~4-fold) in
leukocyte vascular adherence and transmigration
compared to Krebsrsquo buffer (299 plusmn 23 and 271 plusmn
26 leukocytesmm2 of tissue respectively plt001
for both values) Nox2ds-tat dose-dependently
attenuated the L-NAME induced effect on
leukocyte adherence (199 plusmn 13 for 5 microM at
plt005 and 83 plusmn 9 for 20 microM plt001) and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
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SREBP2 activation of NLRP3
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107 DOI 101016jyjmcc201309007
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R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
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DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
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101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
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triumphs and pitfalls Antioxidants amp Redox
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101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
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phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
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249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
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10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
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Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
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R M (1994) Role of the membrane cortex
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33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
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101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
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Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
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Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 219
332 Nox2ds-tat prevented the decrease in
blood NO levels
In saline controls blood NO levels steadily
decreased in the IR limb throughout the
reperfusion period leading to significantly lower
blood NO bioavailability (90 nM below sham) as
early as 20 min into the reperfusion period
(Figure 6 Panel A) By contrast blood NO in the
IR hindlimb of Nox2ds-tat (41 mgkg ~20 μM
in blood) treated animals did not significantly
differ from sham hindlimb values during the
reperfusion period While a linear trend close to
sham values was observed the Nox2ds-tat treated
grouprsquos blood NO levels did rise to 37 nM above
sham values at the end of reperfusion (Figure 6
Panel B) The relative difference in blood NO
levels between IR and sham limbs in Nox2ds-tat
treated animals versus saline controls illustrate
that the Nox2ds-tat treated animals exhibited a
significant increase in the IR induced effect on
NO release by 127 nM at the end of reperfusion
(Figure 6 Panel C)
34 L-NAME induced Leukocyte endothelial
interactions
341 Intravital Microscopy
There was no significant difference among
initial baseline values in leukocyte rolling
adherence and transmigration among all study
groups In all parameters the Krebsrsquo buffer group
did not significantly change from baseline L-
NAME (50 microM) significantly increased leukocyte
rolling adherence and transmigration when
compared to Krebsrsquo at T=120 min by 82 plusmn 11
(plt001) 22 plusmn 5 (plt001) and 21 plusmn 4 (plt001)
respectively (Table 2) Nox2ds-tat (5 microM) did not
significantly attenuate L-NAME induced
leukocyte-endothelial interactions except for final
transmigration (plt005) By contrast Nox2ds-tat
(20 microM) significantly decreased the L-NAME
induced effect by 3-5 fold on all parameters
(plt001) (Table 2)
Table 2 The comparision of initial and final (120 min of reperfusion) leukocyte rolling adherence and transmigration
between Krebsrsquo L-NAME and L-NAME + Nox2ds-tat (5 μM 20 μM) groups in rat mesenteric venules
Intravital microscopy Parameters
Krebsrsquo
(n=6)
L-NAME
(n=5)
L-NAME +
Nox2ds-tat 5 μM
(n=4)
L-NAME +
Nox2ds-tat 20 μM
(n=6)
Initial Rolling (numbermin) 8plusmn3 11plusmn3 13plusmn3 8plusmn2
Final Rolling (numbermin) 16plusmn5 82plusmn11
72plusmn13 16plusmn6++
Initial Adherence (number100um) 2plusmn2 1plusmn0 3plusmn0 1plusmn1
Final Adherence (number100um) 4plusmn2 22plusmn5 17plusmn1 8plusmn4
Initial Transmigration (number) 1plusmn0 1plusmn0 1plusmn0 1plusmn0
Final Transmigration (number) 1plusmn0 21plusmn4 9plusmn1
4plusmn2
Plt005 Plt001 compared to Krebsrsquo groupPlt005
Plt001 compared to L-NAME
++Plt001 compared to L-
NAME + Nox2ds-tat 5 μM
342 HampE Staining
The Krebsrsquocontrol group exhibited only 73 plusmn
33 and 62 plusmn 30 leukocytesmm2 of mesenteric
tissue These values represent basal numbers of
leukocyte adherence and transmigration in this
tissue bed respectively L-NAME treatment
exhibited a marked increase (~4-fold) in
leukocyte vascular adherence and transmigration
compared to Krebsrsquo buffer (299 plusmn 23 and 271 plusmn
26 leukocytesmm2 of tissue respectively plt001
for both values) Nox2ds-tat dose-dependently
attenuated the L-NAME induced effect on
leukocyte adherence (199 plusmn 13 for 5 microM at
plt005 and 83 plusmn 9 for 20 microM plt001) and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
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G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
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101161CIRCULATIONAHA113002714
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M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
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107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
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17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
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1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
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Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
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101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
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triumphs and pitfalls Antioxidants amp Redox
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101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
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10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
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phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
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249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
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105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
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10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
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ischemiareperfusion injury Cardiovascular
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101111j1527-34662005tb00170x
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Mechanisms for the role of
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and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
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R M (1994) Role of the membrane cortex
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33 Manceur A Wu A amp Audet J (2007)
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37 Palaniyandi S S Ferreira J C B Brum P
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(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 220
transmigration (134 plusmn 17 for 5 microM and 55 plusmn 9 for
20 microM plt001 for both values) It should be
noted that the 20 μM dose diminished adherence
and transmigration down to initial values
(Figure 7 Panel A-B)
Figure 7 Panel A HampE stain of mesenteric tissue with representative view (20x) of leukocyte adherence and
transmigration When compared to Krebsrsquo control buffer L-NAME (50 microM) treated tissue displayed a noticeable
increase in leukocyte endothelium adherence and transmigration to the injured site Nox2ds-tat (20 microM) treated tissue
visibly decreased both adherence and transmigrated of leukocytes when compared to L-NAME Arrowhead indicates
adherence and arrow indicate transmigration (20x scale bar 20 microm) Panel B Graph of leukocyte adherence and
transmigration as determined by HampE staining of representative rat mesenteric tissue When compared to Krebsrsquo
control buffer L-NAME treated tissue displayed a significant increase in leukocyte endothelium adherence and
transmigration to injured site (plt001 from Krebsrsquo) Nox2ds-tat dose dependently decreased leukocyte adherence
and transmigration compared to L-NAME treated tissue (plt005 plt001 from to L-NAME) There was also a
significant difference in adherence between the 5 microM and 20 microM doses (++
plt001 vs Nox2ds-tat 5 microM)
A
B
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
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Myocardial reperfusion injury New England
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Myocardial ischemia-reperfusion injury a
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101172JCI62874
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Pathophysiological Roles of NADPH
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101161CIRCRESAHA107162800
6 Duda M Konior A Klemenska E amp
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101016jyjmcc200610014
7 Fabian R H Perez-Polo J R amp Kent T
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Michael L H Youker K A Bressler R
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101089ars20135605
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Activation by Angiotensin II ROLE OF THE
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101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
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Basic Research in Cardiology 106(2) 273-
286 DOI 101007s00395-010-0146-8
14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
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phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
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101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
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101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
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Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
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101111j1582-4934201001174x
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38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 221
4 Discussion
41 Summary of Findings
The major findings of this study are as
follows 1 Nox2ds-tat attenuated PMA-induced
leukocyte SO release up to 37 plusmn 7 (80 μM)
compared to untreated controls (plt005) 2 In
isolated perfused hearts Nox2ds-tat attenuated
infarct sizearea-at-risk by 30 plusmn 4 (5 μM) 15 plusmn
14 (10 μM) 23 plusmn 2 (40 μM) and 19 plusmn 16
(80 μM) when compared to control IR hearts that
had an infarct sizearea-at-risk of 46 plusmn 2 (all
doses plt001 vs IR control) 3 Nox2ds-tat
increased post-reperfused LVDP to 47 plusmn 7 (5
μM) 69 plusmn 14 (10 μM plt005) 68 plusmn 7 (40
μM plt001) and 77 plusmn 7 (80 μM plt005) when
compared to control IR hearts that were 46 plusmn 6
4 Nox2ds-tat significantly decreased post-
reperfused (45 min) blood H2O2 levels by 14 μM
below saline controls (plt001) in hindlimb IR 5
Nox2ds-tat significantly increased post-
reperfused (45 min) blood NO by 127 nM above
saline controls (plt001) in hindlimb IR 6
Nox2ds-tat (20 μM) significantly attenuated L-
NAME induced leukocyte-endothelial
interactions in rat mesenteric venules by 3-to-5
fold and reduced leukocyte rolling adherence
and transmigration back to the initial baseline
values recorded These results suggest that
Nox2ds-tat exerted post-reperfused
cardioprotective and anti-inflammatory effects
possibly by inhibiting ROS release generated by
Nox2
42 Relevance of Study Findings
421 Cardiac Function
Although there was a positive correlation
between the concentration of Nox2ds-tat and
restoration of LVDP the increase in LVDP in the
80 microM dose was primarily related to an increase
in LVESP as opposed to a decrease in LVEDP
which was seen in the 10 μM and 40 microM doses
and also observed in our previous studies [17 24]
This point is significant since this finding is
unprecedented in our ex vivo isolated perfused rat
model exposed to 30 min of ischemia which is
typically characterized by an abrupt increase in
LVEDP (eg 90mmHg) within the first few min
of reperfusion and only relaxing to ~60mmHg
by the end of the reperfusion time period (45 min)
in control IR hearts As seen in the 10 μM and
40 microM doses the drug was able to decrease
LVEDP (while maintaining a similar or slightly
elevated LVESP) and thus increase the LVDP
when compared to control IR hearts The
increase in LVESP in the 80 microM dose suggests
that the heart was able to produce a contraction
strong enough to compensate for the increased
LVEDP We believe that at this high dose (80
microM) it caused a shift in the cardiac contractility
parameters wherein there is both an increase in
the LVEDP and LVESP when compared to the
10 μM and 40 microM doses (Table 1) It should be
noted that the increase in post-reperfused LVDP
for the 80 microM dose when compared to control IR
hearts is due to the LVESP increasing more than
LVEDP We speculate that these results are
indicative of reduced Nox2-generated ROS
release which in turn attenuated ROS-induced
dysfunction of the cardiomyocyte sarcoplasmic
reticulum This putative effect would
subsequently improve Ca2+
handling in the post-
reperfused cardiomyocyte which would result in
improved cardiac contractility [2] As seen in
previous studies it should be emphasized that the
cardioprotective effect of agents such as apocynin
typically show a reduction in LVEDP to
ultimately manifest their improvement in cardiac
function as represented by the increase in LVDP
[17]
422 Infarct Size
Similar to the cardiac function data the
lowest dose tested (5 μM) corresponded with the
largest infarct size (30) among the Nox2ds-tat
treated groups however this dose was still
significantly different from IR control (46)
However this dose resulted in a significantly
larger infarct size than the 10 μM (plt001) and
80 microM (plt005) doses The 10 μM 40 microM and
80 microM doses all showed a significant reduction in
infarct size (15 -23) when compared to
control (46) We speculate that the
improvement in LVDP is related to the
attenuation of SO release in cardiac tissue seen
with all three doses (10 μM 40 microM and 80 microM)
This suggests that the degree of cell death is
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
References
1 Yellon D M amp Hausenloy D J (2007)
Myocardial reperfusion injury New England
Journal of Medicine 357(11) 1121-1135
DOI 101056NEJMra071667
2 Hausenloy D J amp Yellon D M (2013)
Myocardial ischemia-reperfusion injury a
neglected therapeutic target Journal of
Clinical Investigation 123(1) 92-100 DOI
101172JCI62874
3 Lefer A M amp Lefer D J (1996) The role
of nitric oxide and cell adhesion molecules
on the microcirculation in ischaemia-
reperfusion Cardiovascular Research 32(4)
743-751 DOI 1010160008-
6363(96)00073-9
4 Ago T Kuroda J Kamouchi M
Sadoshima J amp Kitazono T (2011)
Pathophysiological Roles of NADPH
OxidaseNox family proteins in the vascular
system-review and perspective Circulation
Journal 75(8) 1791-1800 DOI
101253circjCJ-11-0388
5 Doughan A K Harrison D G amp Dikalov
S I (2008) Molecular Mechanisms of
Angiotensin IIndashMediated Mitochondrial
Dysfunction Linking Mitochondrial
Oxidative Damage and Vascular Endothelial
Dysfunction Circulation research 102(4)
488-496 DOI
101161CIRCRESAHA107162800
6 Duda M Konior A Klemenska E amp
Beręsewicz A (2007) Preconditioning
protects endothelium by preventing ET-1-
induced activation of NADPH oxidase and
xanthine oxidase in post-ischemic heart
Journal of Molecular and Cellular
Cardiology 42(2) 400-410 DOI
101016jyjmcc200610014
7 Fabian R H Perez-Polo J R amp Kent T
A (2008) Perivascular nitric oxide and
superoxide in neonatal cerebral hypoxia-
ischemia American Journal of Physiology-
Heart and Circulatory Physiology 295(4)
H1809-H1814 DOI
101152ajpheart003012007
8 Frangogiannis N G Lindsey M L
Michael L H Youker K A Bressler R
B Mendoza L H amp Entman M L
(1998) Resident cardiac mast cells
degranulate and release preformed TNF-α
initiating the cytokine cascade in
experimental canine myocardial
ischemiareperfusion Circulation 98(7)
699-710 DOI 10116101CIR987699
9 Nolly M B Caldiz C I Yeves A M
Villa-Abrille M C Morgan P E
Mondaca N A amp Ennis I L (2014)
The signaling pathway for aldosterone-
induced mitochondrial production of
superoxide anion in the myocardium
Journal of Molecular and Cellular
Cardiology 67 60-68 DOI
101016jyjmcc201312004
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 225
10 Kleniewska P Piechota A Skibska B amp
Gorąca A (2012) The NADPH oxidase
family and its inhibitors Archivum
immunologiae et therapiae experimentalis
60(4) 277-294 DOI 101007s00005-012-
0176-z
11 Laurindo F R Araujo T L amp Abrahao T
B (2014) Nox NADPH oxidases and the
endoplasmic reticulum Antioxidants amp
Redox Signaling 20(17) 2755-2775 DOI
101089ars20135605
12 Li J M amp Shah A M (2003) Mechanism
of Endothelial Cell NADPH Oxidase
Activation by Angiotensin II ROLE OF THE
p47 phox SUBUNIT Journal of Biological
Chemistry 278(14) 12094-12100 DOI
101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
attenuated neurohumoral excitation in heart
failure a role for superoxide and nitric oxide
Basic Research in Cardiology 106(2) 273-
286 DOI 101007s00395-010-0146-8
14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 226
phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
Biochemistry 46(51) 14771-14781 DOI
101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
Immunology 33(5) 1328-1333 DOI
101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
inhibition attenuates myocardial infarction
induced heart failure and is associated with a
reduction of fibrosis and pro‐inflammatory
responses Journal of Cellular and
Molecular Medicine 15(8) 1769-1777 DOI
101111j1582-4934201001174x
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 222
positively correlated with SO release from
cardiac tissue and negatively correlated with
restoration of post-reperfused cardiac function
findings which are consistent with our previous
studies [17 24 25]
423 Leukocyte-Endothelial Interactions
4231 Intravital Microscopy and HampE
Staining
It is well known that SO can reduce NO
bioavailability and form peroxynitrite anion [29]
Therefore inhibiting Nox2 ROS generation
would inhibit the quenching effect that SO exerts
on NO bioavailability This would in turn
increase NO bioavailability which would reduce
the upregulation of vascular adhesion molecules
responsible for sequestering leukocytes to the
vascular endothelium and initiating
transmigration into tissue [3] As expected we
observed a dose-dependent effect on leukocyte
endothelial interactions in rat mesenteric venules
Similar to our ex vivo isolated heart model the
effect of the 5 μM dose in this in vivo assay was
not different from the L-NAME control whereas
the 20 microM dose attenuated the L-NAME induced
effect 3-5 fold (Table 2) This data suggests that
L-NAME via inhibiting NO production induced
an inflammatory response ultimately increasing
Nox2 activity Moreover HampE staining of
mesenteric tissue isolated at the end of the
intravital microscopy procedure corroborated the
real-time data More leukocytes were observed in
mesenteric venules in the presence of L-NAME
an effect that was dose-dependently inhibited by
Nox2ds-tat (Figure 7 Panel A-B) These findings
are consistent with previous studies [24 27]
4232 PMA-induced SO Release
Only the 80 microM dose showed a significant
decrease in PMA-induced SO release from PMNs
when compared to controls (Figure 3) We
speculate the blunted effect of Nox2ds-tat (10 μM
and 40 microM) on PMA-induced SO release from
PMNs was due to restrictions within the
membrane of neutrophils First research has
shown the membrane cortex of neutrophils
becomes rigid when exposed to mechanical stress
during in vitro assays making the surface
resistant to deformation [30] Moreover this
mechanical stress results in the reorganization
and polymerization of a neutrophilrsquos cytoskeleton
and the possible disruption of associated
transporters and second messenger systems [31
32] Thus in vitro cell transport mechanisms for
TAT-internalization may not be equivalent to ex
vivo models which maintain a more intact
transport system perhaps due to less systematic
stress upon the neutrophils Second
hematopoietic stem cells such as PMNs are more
resistant to TAT-internalization especially when
compared to cargo sequences conjugated with a
smaller and more permeable cell penetrating
peptide such as myristic acid [33 34] In final
support of PMN resistance to TAT-conjugated
peptide internalization Rey et al [19] found
similar results in PMA-induced human PMN SO
release when comparing the efficacy of Nox2ds-
tat in a dose range similar to our own
424 Hindlimb IR
4241 Blood H2O2 release
We chose to use 41 mg of Nox2ds-tat per kg
of tissue (~20 microM in blood) because this was in
the mid-dose range of the effective
concentrations that showed a significant decrease
in LVEDP (in our ex vivo cardiac function data)
Compared to lose dose Nox2ds-tat (5microM) this
dose reduced post-reperfused blood H2O2 levels
back to sham control values by the end of the
experiment (45 min reperfusion) By contrast
saline controls maintained post-reperfused H2O2
blood levels above sham control values
throughout the reperfusion period (45 min) The
data from this assay suggests Nox2 activation and
ROS production during reperfusion is the
principal source of ROS in this IR model and
mechanistically may explain the attenuation of
infarct size observed in our isolated perfused
heart model This finding is consistent with our
previous study [17] Moreover the hindlimb IR
assay further corroborates the speculation that
Nox2 expressed in cardiovascular tissue is the
principle source of ROS during reperfusion
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
References
1 Yellon D M amp Hausenloy D J (2007)
Myocardial reperfusion injury New England
Journal of Medicine 357(11) 1121-1135
DOI 101056NEJMra071667
2 Hausenloy D J amp Yellon D M (2013)
Myocardial ischemia-reperfusion injury a
neglected therapeutic target Journal of
Clinical Investigation 123(1) 92-100 DOI
101172JCI62874
3 Lefer A M amp Lefer D J (1996) The role
of nitric oxide and cell adhesion molecules
on the microcirculation in ischaemia-
reperfusion Cardiovascular Research 32(4)
743-751 DOI 1010160008-
6363(96)00073-9
4 Ago T Kuroda J Kamouchi M
Sadoshima J amp Kitazono T (2011)
Pathophysiological Roles of NADPH
OxidaseNox family proteins in the vascular
system-review and perspective Circulation
Journal 75(8) 1791-1800 DOI
101253circjCJ-11-0388
5 Doughan A K Harrison D G amp Dikalov
S I (2008) Molecular Mechanisms of
Angiotensin IIndashMediated Mitochondrial
Dysfunction Linking Mitochondrial
Oxidative Damage and Vascular Endothelial
Dysfunction Circulation research 102(4)
488-496 DOI
101161CIRCRESAHA107162800
6 Duda M Konior A Klemenska E amp
Beręsewicz A (2007) Preconditioning
protects endothelium by preventing ET-1-
induced activation of NADPH oxidase and
xanthine oxidase in post-ischemic heart
Journal of Molecular and Cellular
Cardiology 42(2) 400-410 DOI
101016jyjmcc200610014
7 Fabian R H Perez-Polo J R amp Kent T
A (2008) Perivascular nitric oxide and
superoxide in neonatal cerebral hypoxia-
ischemia American Journal of Physiology-
Heart and Circulatory Physiology 295(4)
H1809-H1814 DOI
101152ajpheart003012007
8 Frangogiannis N G Lindsey M L
Michael L H Youker K A Bressler R
B Mendoza L H amp Entman M L
(1998) Resident cardiac mast cells
degranulate and release preformed TNF-α
initiating the cytokine cascade in
experimental canine myocardial
ischemiareperfusion Circulation 98(7)
699-710 DOI 10116101CIR987699
9 Nolly M B Caldiz C I Yeves A M
Villa-Abrille M C Morgan P E
Mondaca N A amp Ennis I L (2014)
The signaling pathway for aldosterone-
induced mitochondrial production of
superoxide anion in the myocardium
Journal of Molecular and Cellular
Cardiology 67 60-68 DOI
101016jyjmcc201312004
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 225
10 Kleniewska P Piechota A Skibska B amp
Gorąca A (2012) The NADPH oxidase
family and its inhibitors Archivum
immunologiae et therapiae experimentalis
60(4) 277-294 DOI 101007s00005-012-
0176-z
11 Laurindo F R Araujo T L amp Abrahao T
B (2014) Nox NADPH oxidases and the
endoplasmic reticulum Antioxidants amp
Redox Signaling 20(17) 2755-2775 DOI
101089ars20135605
12 Li J M amp Shah A M (2003) Mechanism
of Endothelial Cell NADPH Oxidase
Activation by Angiotensin II ROLE OF THE
p47 phox SUBUNIT Journal of Biological
Chemistry 278(14) 12094-12100 DOI
101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
attenuated neurohumoral excitation in heart
failure a role for superoxide and nitric oxide
Basic Research in Cardiology 106(2) 273-
286 DOI 101007s00395-010-0146-8
14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 226
phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
Biochemistry 46(51) 14771-14781 DOI
101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
Immunology 33(5) 1328-1333 DOI
101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
inhibition attenuates myocardial infarction
induced heart failure and is associated with a
reduction of fibrosis and pro‐inflammatory
responses Journal of Cellular and
Molecular Medicine 15(8) 1769-1777 DOI
101111j1582-4934201001174x
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 223
4242 Blood NO release
Nox2ds-tat completely reversed the effect of
IR on NO bioavailability which fell to 90 nM
below the sham limb in saline treated groups
(Figure 6 Panel A) Administration of Nox2ds-
tat maintained blood NO levels that did not
significantly differ from the sham limb however
a total increase of 37 nM above sham hindlimb
values was observed (Figure 6 Panel B)
Ultimately the treatment significantly increased
blood NO by 127 nM above saline controls at the
end of the reperfusion period (45 min) (Figure 6
Panel C) This effect was most likely the result of
inhibiting Nox2-induced SO production and the
subsequent attenuation of the quenching effect
that ROS has on NO bioavailability [3] This
effect would also explain the decrease in
leukocyte-endothelial interactions in mesenteric
venules and the reduction of infarct size in the
isolated perfused rat heart model
43 Role of Nox2 in IR injury
Nox2 activation during reperfusion occurs as
a result of proinflammatory cytokine receptor
activation in leukocytes and cardiovascular tissue
(eg TNF-α and NF-κB) accompanied by
chemokine receptor upregulation and recruitment
of inflammatory mediators Since the majority of
chemokine receptors integrate G protein coupled
receptor kinases this inflammation results in a
cascade of events which culminate in PKC
activation and a subsequent upregulation of Nox2
[12 13 35] Activation of Nox2 promotes
endothelial dysfunction by diminishing the
bioavailability of NO and propagating eNOS
uncoupling via BH4 depletion this will shift the
product profile of eNOS to primarily producing
SO instead of cardioprotective NO [29]
Moreover Nox2 activation on organelle
membranes will produce intracellular SO which
leads to uncoupling of the mitochondrial electron
transport chain and additional ROS production
[11] The oxidation of xanthine dehydrogenase to
xanthine oxidase will also favor the profile of this
enzyme to produce SO [1 2] Collectively the
activity of Nox2 will lead to the induction of SO
generation by the aforementioned sources and
propagate the deleterious cycle of SO production
and NO depletion characteristic of IR injury
The role that Nox2 plays in myocardial IR
injury is crucial to understand since general
antioxidants such as vitamin-C and vitamin-E
have proven to be ineffective in the attenuation of
SO injury in clinical myocardial IR in part
because they have limited access in targeting the
primary source generating this oxidative stress [1
2] Identifying a key source of ROS generation
could be essential since more than 50yrs have
passed since reperfusion injury has been
identified as a major clinical problem affecting
MI patients and there is still no pharmacological
treatment to attenuate myocardial reperfusion
injury [36] IR injury is a multifactorial process
that is initiated by ROS generation which in turn
causes additional injury to adjacent cells and
upregulation of inflammatory cytokines which in
turn attract more leukocytes to the area of injury
ultimately reinforcing a cascade of events that
exacerbate the original ischemic injury
Attenuating the primary sources of oxidative
stress during IR injury will inhibit downstream
effects such as histamine release from mast cells
and TGF-β secretion from injured cells these
events lead to the activation of NF-κB and
programmed cell death processes which occur on
a chronic basis during the weeks following a MI
[37] Inhibiting the generation of ROS during IR
injury in an early effective and selective manner
will attenuate the downstream processes which
result from SO release and more importantly
minimize the chronic administration of
pharmaceuticals aimed to mitigate the symptoms
associated with cardiac remodeling following a
MI
44 Future Studies
Nox2ds-tat is a putative inhibitor of Nox2
the primary NADPH isoform in leukocytes and
the cardiovascular system NADPH oxidases are
unique in their ability to perpetuate other sources
of oxidative stress lending to why this study
focused specifically on this peptide However it
would be of great interest to compare the effects
of Nox2ds-tat alone versus the effects of this
peptide in conjunction with inhibitors of the other
three enzymatic sources of oxidative stress during
IR injury (ie uncoupling of the mitochondrial
electron transport chain uncoupled eNOS and
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
References
1 Yellon D M amp Hausenloy D J (2007)
Myocardial reperfusion injury New England
Journal of Medicine 357(11) 1121-1135
DOI 101056NEJMra071667
2 Hausenloy D J amp Yellon D M (2013)
Myocardial ischemia-reperfusion injury a
neglected therapeutic target Journal of
Clinical Investigation 123(1) 92-100 DOI
101172JCI62874
3 Lefer A M amp Lefer D J (1996) The role
of nitric oxide and cell adhesion molecules
on the microcirculation in ischaemia-
reperfusion Cardiovascular Research 32(4)
743-751 DOI 1010160008-
6363(96)00073-9
4 Ago T Kuroda J Kamouchi M
Sadoshima J amp Kitazono T (2011)
Pathophysiological Roles of NADPH
OxidaseNox family proteins in the vascular
system-review and perspective Circulation
Journal 75(8) 1791-1800 DOI
101253circjCJ-11-0388
5 Doughan A K Harrison D G amp Dikalov
S I (2008) Molecular Mechanisms of
Angiotensin IIndashMediated Mitochondrial
Dysfunction Linking Mitochondrial
Oxidative Damage and Vascular Endothelial
Dysfunction Circulation research 102(4)
488-496 DOI
101161CIRCRESAHA107162800
6 Duda M Konior A Klemenska E amp
Beręsewicz A (2007) Preconditioning
protects endothelium by preventing ET-1-
induced activation of NADPH oxidase and
xanthine oxidase in post-ischemic heart
Journal of Molecular and Cellular
Cardiology 42(2) 400-410 DOI
101016jyjmcc200610014
7 Fabian R H Perez-Polo J R amp Kent T
A (2008) Perivascular nitric oxide and
superoxide in neonatal cerebral hypoxia-
ischemia American Journal of Physiology-
Heart and Circulatory Physiology 295(4)
H1809-H1814 DOI
101152ajpheart003012007
8 Frangogiannis N G Lindsey M L
Michael L H Youker K A Bressler R
B Mendoza L H amp Entman M L
(1998) Resident cardiac mast cells
degranulate and release preformed TNF-α
initiating the cytokine cascade in
experimental canine myocardial
ischemiareperfusion Circulation 98(7)
699-710 DOI 10116101CIR987699
9 Nolly M B Caldiz C I Yeves A M
Villa-Abrille M C Morgan P E
Mondaca N A amp Ennis I L (2014)
The signaling pathway for aldosterone-
induced mitochondrial production of
superoxide anion in the myocardium
Journal of Molecular and Cellular
Cardiology 67 60-68 DOI
101016jyjmcc201312004
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 225
10 Kleniewska P Piechota A Skibska B amp
Gorąca A (2012) The NADPH oxidase
family and its inhibitors Archivum
immunologiae et therapiae experimentalis
60(4) 277-294 DOI 101007s00005-012-
0176-z
11 Laurindo F R Araujo T L amp Abrahao T
B (2014) Nox NADPH oxidases and the
endoplasmic reticulum Antioxidants amp
Redox Signaling 20(17) 2755-2775 DOI
101089ars20135605
12 Li J M amp Shah A M (2003) Mechanism
of Endothelial Cell NADPH Oxidase
Activation by Angiotensin II ROLE OF THE
p47 phox SUBUNIT Journal of Biological
Chemistry 278(14) 12094-12100 DOI
101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
attenuated neurohumoral excitation in heart
failure a role for superoxide and nitric oxide
Basic Research in Cardiology 106(2) 273-
286 DOI 101007s00395-010-0146-8
14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 226
phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
Biochemistry 46(51) 14771-14781 DOI
101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
Immunology 33(5) 1328-1333 DOI
101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
inhibition attenuates myocardial infarction
induced heart failure and is associated with a
reduction of fibrosis and pro‐inflammatory
responses Journal of Cellular and
Molecular Medicine 15(8) 1769-1777 DOI
101111j1582-4934201001174x
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 224
xanthine oxidase formation) Our results for the 5
μM dose of Nox2ds-tat did not show significant
recovery of post-reperfused cardiac function but
one may speculate that a low dose of Nox2ds-tat
in conjunction with another selective peptide
inhibitor of one of the other sources of IR
induced ROS may render significant results For
example SS-31 a targeted mitochondrial
antioxidant peptide has been shown to dose-
dependently improve post-reperfused LVDP and
reduce infarct size in rat hearts subjected to I (30
min)R (45 min) in a manner similar to Nox2ds-
tat [38 39] While a low dose of these peptides
administered alone did not yield significant
results a combination of Nox2ds-tat (5 μM) with
SS-31 (10 μM) synergistically improved post-
reperfused LVDP by 63 plusmn 11 of initial values
when compared to Nox2ds-tat (5 μM) alone
(Table 1) SS-31 alone (10 μM) or control
untreated IR hearts that recovered to 47 plusmn 7
25 plusmn 1 and 46 plusmn 6 (Table 1) respectively
[38] These results are promising and lead to an
important direction for future studies because
they imply that lower doses of selective ROS
inhibitory peptides may complement one another
and be used to synergistically reduce myocardial
dysfunction during IR injury The use of lower
doses can possibly minimize adverse drug
reactions and immunological responses in target
patient populations
References
1 Yellon D M amp Hausenloy D J (2007)
Myocardial reperfusion injury New England
Journal of Medicine 357(11) 1121-1135
DOI 101056NEJMra071667
2 Hausenloy D J amp Yellon D M (2013)
Myocardial ischemia-reperfusion injury a
neglected therapeutic target Journal of
Clinical Investigation 123(1) 92-100 DOI
101172JCI62874
3 Lefer A M amp Lefer D J (1996) The role
of nitric oxide and cell adhesion molecules
on the microcirculation in ischaemia-
reperfusion Cardiovascular Research 32(4)
743-751 DOI 1010160008-
6363(96)00073-9
4 Ago T Kuroda J Kamouchi M
Sadoshima J amp Kitazono T (2011)
Pathophysiological Roles of NADPH
OxidaseNox family proteins in the vascular
system-review and perspective Circulation
Journal 75(8) 1791-1800 DOI
101253circjCJ-11-0388
5 Doughan A K Harrison D G amp Dikalov
S I (2008) Molecular Mechanisms of
Angiotensin IIndashMediated Mitochondrial
Dysfunction Linking Mitochondrial
Oxidative Damage and Vascular Endothelial
Dysfunction Circulation research 102(4)
488-496 DOI
101161CIRCRESAHA107162800
6 Duda M Konior A Klemenska E amp
Beręsewicz A (2007) Preconditioning
protects endothelium by preventing ET-1-
induced activation of NADPH oxidase and
xanthine oxidase in post-ischemic heart
Journal of Molecular and Cellular
Cardiology 42(2) 400-410 DOI
101016jyjmcc200610014
7 Fabian R H Perez-Polo J R amp Kent T
A (2008) Perivascular nitric oxide and
superoxide in neonatal cerebral hypoxia-
ischemia American Journal of Physiology-
Heart and Circulatory Physiology 295(4)
H1809-H1814 DOI
101152ajpheart003012007
8 Frangogiannis N G Lindsey M L
Michael L H Youker K A Bressler R
B Mendoza L H amp Entman M L
(1998) Resident cardiac mast cells
degranulate and release preformed TNF-α
initiating the cytokine cascade in
experimental canine myocardial
ischemiareperfusion Circulation 98(7)
699-710 DOI 10116101CIR987699
9 Nolly M B Caldiz C I Yeves A M
Villa-Abrille M C Morgan P E
Mondaca N A amp Ennis I L (2014)
The signaling pathway for aldosterone-
induced mitochondrial production of
superoxide anion in the myocardium
Journal of Molecular and Cellular
Cardiology 67 60-68 DOI
101016jyjmcc201312004
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 225
10 Kleniewska P Piechota A Skibska B amp
Gorąca A (2012) The NADPH oxidase
family and its inhibitors Archivum
immunologiae et therapiae experimentalis
60(4) 277-294 DOI 101007s00005-012-
0176-z
11 Laurindo F R Araujo T L amp Abrahao T
B (2014) Nox NADPH oxidases and the
endoplasmic reticulum Antioxidants amp
Redox Signaling 20(17) 2755-2775 DOI
101089ars20135605
12 Li J M amp Shah A M (2003) Mechanism
of Endothelial Cell NADPH Oxidase
Activation by Angiotensin II ROLE OF THE
p47 phox SUBUNIT Journal of Biological
Chemistry 278(14) 12094-12100 DOI
101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
attenuated neurohumoral excitation in heart
failure a role for superoxide and nitric oxide
Basic Research in Cardiology 106(2) 273-
286 DOI 101007s00395-010-0146-8
14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 226
phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
Biochemistry 46(51) 14771-14781 DOI
101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
Immunology 33(5) 1328-1333 DOI
101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
inhibition attenuates myocardial infarction
induced heart failure and is associated with a
reduction of fibrosis and pro‐inflammatory
responses Journal of Cellular and
Molecular Medicine 15(8) 1769-1777 DOI
101111j1582-4934201001174x
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 225
10 Kleniewska P Piechota A Skibska B amp
Gorąca A (2012) The NADPH oxidase
family and its inhibitors Archivum
immunologiae et therapiae experimentalis
60(4) 277-294 DOI 101007s00005-012-
0176-z
11 Laurindo F R Araujo T L amp Abrahao T
B (2014) Nox NADPH oxidases and the
endoplasmic reticulum Antioxidants amp
Redox Signaling 20(17) 2755-2775 DOI
101089ars20135605
12 Li J M amp Shah A M (2003) Mechanism
of Endothelial Cell NADPH Oxidase
Activation by Angiotensin II ROLE OF THE
p47 phox SUBUNIT Journal of Biological
Chemistry 278(14) 12094-12100 DOI
101074jbcM209793200
13 Guggilam A Cardinale J P Mariappan
N Sriramula S Haque M amp Francis J
(2011) Central TNF inhibition results in
attenuated neurohumoral excitation in heart
failure a role for superoxide and nitric oxide
Basic Research in Cardiology 106(2) 273-
286 DOI 101007s00395-010-0146-8
14 Xiao H Lu M Lin T Y Chen Z Chen
G Wang W C amp Sun W (2013)
SREBP2 activation of NLRP3
inflammasome in endothelium mediates
hemodynamic-induced atherosclerosis
susceptibility Circulation
CIRCULATIONAHA-113 DOI
101161CIRCULATIONAHA113002714
15 Braunersreuther V Montecucco F Ashri
M Pelli G Galan K Frias M amp Mach
F (2013) Role of NADPH oxidase isoforms
NOX1 NOX2 and NOX4 in myocardial
ischemiareperfusion injury Journal of
Molecular and Cellular Cardiology 64 99-
107 DOI 101016jyjmcc201309007
16 Somasuntharam I Boopathy A V Khan
R S Martinez M D Brown M E
Murthy N amp Davis M E (2013) Delivery
of Nox2-NADPH oxidase siRNA with
polyketal nanoparticles for improving
cardiac function following myocardial
infarction Biomaterials 34(31) 7790-7798
DOI 101016jbiomaterials201306051
17 Chen Q Parker CW Devine I Ondrasik
R Habtamu T Bartol K D Casey B
amp Young L (2016) Apocynin Exerts
Dose-Dependent Cardioprotective Effects by
Attenuating Reactive Oxygen Species in
IschemiaReperfusion Cardiovascular
Pharmacology Open Access 2016 DOI
1041762329-66071000176
18 Johnson D K Schillinger K J Kwait D
M Hughes C V McNamara E J Ishmael
F amp Santhanam L (2002) Inhibition of
NADPH oxidase activation in endothelial
cells by ortho-methoxy-substituted catechols
Endothelium 9(3) 191-203 DOI
10108010623320213638
19 Rey F E Cifuentes M E Kiarash A
Quinn M T amp Pagano P J (2001) Novel
competitive inhibitor of NAD(P)H oxidase
assembly attenuates vascular O2minus and
systolic blood pressure in mice Circulation
Research 89(5) 408-414 DOI
101161hh1701096037
20 Csaacutenyi G Cifuentes-Pagano E Al
Ghouleh I Ranayhossaini D J Egantildea L
Lopes L R amp Pagano P J (2011)
Nox2 B-loop peptide Nox2ds specifically
inhibits the NADPH oxidase Nox2 Free
Radical Biology and Medicine 51(6) 1116-
1125 DOI
101016jfreeradbiomed201104025
21 Cifuentes-Pagano E Meijles D N amp
Pagano P J (2014) The quest for selective
nox inhibitors and therapeutics challenges
triumphs and pitfalls Antioxidants amp Redox
Signaling 20(17) 2741-2754 DOI
101089ars20135620
22 Jacobson G M Dourron H M Liu J
Carretero O A Reddy D J Andrzejewski
T amp Pagano P J (2003) Novel NAD(P)H
oxidase inhibitor suppresses angioplasty-
induced superoxide and neointimal
hyperplasia of rat carotid artery Circulation
Research 92(6) 637-643 DOI
10116101RES0000063423946458A
23 Perkins K A Bartol K Chen Q
Feinstein E amp Young L (2011)
Myristoylation of protein kinase C beta
IIzeta peptide inhibitors or caveolin-1
peptide facilitates rapid attenuation of
phorbol 12-myristate 13-acetate (PMA) or N-
formyl-L-methionyl-L-leucyl-L-
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 226
phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
transport of peptides into living cells
Biochemistry 46(51) 14771-14781 DOI
101021bi701295k
35 Soriano S F Serrano A Hernanz‐Falcoacuten
P de Ana A M Monterrubio M
Martiacutenez‐A C amp Mellado M (2003)
Chemokines integrate JAKSTAT and G‐protein pathways during chemotaxis and
calcium flux responses European Journal of
Immunology 33(5) 1328-1333 DOI
101002eji200323897
36 Jennings R Aacute Sommers H Aacute Smyth G
A Flack H A amp Linn H (1960)
Myocardial necrosis induced by temporary
occlusion of a coronary artery in the dog
Archives of Pathology 70 68-78
37 Palaniyandi S S Ferreira J C B Brum P
C amp Mochly‐Rosen D (2011) PKCβII
inhibition attenuates myocardial infarction
induced heart failure and is associated with a
reduction of fibrosis and pro‐inflammatory
responses Journal of Cellular and
Molecular Medicine 15(8) 1769-1777 DOI
101111j1582-4934201001174x
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 226
phenylalanine (fMLP) activated leukocyte
superoxide release Proceedings of the 22nd
American Peptide Symposium Michal Lebl
(Editor) American Peptide Society 288-289
24 Blakeman N Chen Q Tolson J Rueter
B Diaz B Casey B amp Weis M T
(2012) Triacsin C a fatty acyl coA
synthetase inhibitor improves cardiac
performance following global ischemia
American Journal of Biomed Sciences 4
249-261 DOI 105099aj120300252
25 Chen Q Kim E E J Elio K Zambrano
C Krass S Teng J C W amp Emrich J
(2010) The role of tetrahydrobiopterin and
dihydrobiopterin in ischemiareperfusion
injury when given at reperfusion Advances
in Pharmacological Sciences vol 2010
Article ID 963914 DOI
1011552010963914
26 Young L H Chen Q amp Weis M T
(2011) Direct Measurement of Hydrogen
Peroxide (H2O2) or Nitric Oxide (NO)
Release A Powerful Tool to Assess Real-
time Free Radical Production in Biological
Models American Journal of Biomed
Sciences 3(1) 40-48 DOI
105099aj110100040
27 Chen Q Rueter B Krass S Zambrano
C Thomas S Prince C amp Young L
(2010) The potential clinical application of
protein kinase C beta II peptide inhibitor or
Gouml 6983 in vascular endothelial dysfunction
Current Topics in Pharmacology 14(4) 11-
24
httpwwwresearchtrendsnettiaabstractas
pin=0ampvn=14amptid=11ampaid=3050amppub=20
10amptype=3
28 Young L H Balin B J amp Weis M T
(2005) Go 6983 a fast acting protein kinase
C inhibitor that attenuates myocardial
ischemiareperfusion injury Cardiovascular
Drug Reviews 23(3) 255-272 DOI
101111j1527-34662005tb00170x
29 Schmidt T S amp Alp N J (2007)
Mechanisms for the role of
tetrahydrobiopterin in endothelial function
and vascular disease Clinical Science
113(2) 47-63 DOI 101042CS20070108
30 Zhelev D V Needham D amp Hochmuth
R M (1994) Role of the membrane cortex
in neutrophil deformation in small pipets
Biophysical Journal 67(2) 696-705 DOI
101016S0006-3495(94)80529-6
31 Zhelev D V amp Hochmuth R M (1995)
Mechanically stimulated cytoskeleton
rearrangement and cortical contraction in
human neutrophils Biophysical Journal
68(5) 2004-2014 DOI 101016S0006-
3495(95)80377-2
32 Mills J W amp Mandel L J (1994)
Cytoskeletal regulation of membrane
transport events The FASEB Journal 8(14)
1161-1165
33 Manceur A Wu A amp Audet J (2007)
Flow cytometric screening of cell-
penetrating peptides for their uptake into
embryonic and adult stem cells Analytical
Biochemistry 364(1) 51-59 DOI
101016jab200702015
34 Nelson A R Borland L Allbritton N L
amp Sims C E (2007) Myristoyl-based
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38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
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W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
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The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30
Am J Biomed Sci 2016 8(3) 208-227 doi 105099aj160300208 copy 2016 by NWPII All rights reserved 227
38 Patel H Walker S Chau W Devine I
Ondrasik R Chen Q amp Young L
(2014) Combinational effects of gp91 ds-tat
and SS-31 in reducing myocardialischemia
reperfusion injury The FASEB Journal 28(1
Supplement) 1080-8
39 Ondrasik R Chen Q Navitsky K Chau
W Lau O S Devine I amp Young L H
(2013) Cardioprotective Effects of Selective
Mitochondrial-Targeted Antioxidants in
Myocardial IschemiaReperfusion (IR)
Injury Proceedings of the 23rd American
Peptide Symposium Michal Lebl (Editor)
American Peptide Society 64-65 DOI
101795223APS2013064
40 Wilkinson B L amp Landreth G E (2006)
The microglial NADPH oxidase complex as
a source of oxidative stress in Alzheimers
disease Journal of Neuroinflammation 3(1)
1 DOI 1011861742-2094-3-30